Genetic loci associated with resistance of soybean to cyst nematode and methods of use

ABSTRACT

Various compositions and methods are provided for identifying and selecting plants with enhanced resistance to soybean cyst nematode (SCN). Further provided are transgenic plants, plant parts, and seed and methods of their use comprising a heterologous polynucleotide operably linked to a promoter active in the plant are provided, as are methods of making such plants and methods of use, wherein said heterologous polynucleotide comprises at least one, or any combination thereof, of Glyma18g2580, Glyma18g2590, Glyma18g2600, Glyma18g2610; and Glyma18g2570 or an active variant or fragment thereof. Expression of the heterologous polynucleotide enhances the resistance of the plant to cyst nematode.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Patent ApplicationSer. No. 61/740,526, filed on Dec. 21, 2012, U.S. Provisional PatentApplication Ser. No. 61/660,387, filed on Jun. 15, 2012, and U.S.Provisional Patent Application Ser. No. 61/671,937, filed Jul. 16, 2012,each of which is hereby incorporated herein in its entirety byreference.

FIELD OF THE INVENTION

The present disclosure relates to compositions and methods useful increating or enhancing cyst nematode resistance in plants.

REFERENCE TO A SEQUENCE LISTING SUBMITTED AS A TEXT FILE VIA EFS-WEB

The official copy of the sequence listing is submitted electronicallyvia EFS-Web as an ASCII formatted sequence listing with a file named430057SEQLIST.txt, created on Feb. 19, 2013, and having a size of 20 KBand is filed concurrently with the specification. The sequence listingcontained in this ASCII formatted document is part of the specificationand is herein incorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

Soybean Cyst Nematode (SCN) is a parasitic pest which has threatenedsoybean production in the U.S. for more than fifty years. SCN resistanceis an economically important trait as infection can substantially reduceyields. Despite this, two primary sources of resistance contribute toelite Pioneer germplasm: Peking and PI88788. Several loci have beenreported to confer SCN resistance, arguably the most important of theserhg1 maps to linkage group G and is comprised of at least two alleles:rhg1 derived from Peking and rhg1-b derived from PI88788.

Cloning of the rhg1 allele was reported previously, and a candidatereceptor-like kinase gene is the subject of a competitor's patentapplications; despite this, no genetic evidence has been provided tosupport these claims. Furthermore, a recent study fine-mapped the rhg1-ballele to a 67-kb region which does not include the rhg1 candidate gene.In light of these reports, the true molecular nature of rhg1 and rhg1-bSCN resistant alleles remains unclear of the same gene and it isuncertain whether rhg1 and rhg1-b are alleles of the same resistant geneor represent two distinct albeit tightly linked genetic loci.

Molecular characterization of these alleles would have importantimplications for soybean cultivar improvement.

SUMMARY

Compositions and methods for identifying and selecting plants withenhanced resistance to soybean cyst nematode (SCN) are provided.

Methods are provided for identifying and/or selecting a soybean plant ora soybean germplasm with enhanced resistance to soybean cyst nematode.In these methods, the presence of at least one marker allele is detectedin the genome of the soybean plant or soybean germplasm; and, a soybeanplant or soybean germplasm with enhanced resistance to cyst nematode isthereby identified and/or selected. The marker allele can include anymarker allele that is associated with a duplication of a region withinthe rhg1 locus which confers enhanced tolerance to soybean cystnematodes.

Further provided are methods of identifying and/or selecting a soybeanplant or a soybean germplasm with enhanced resistance to soybean cystnematode employing quantitative PCR or other quantitative technique ofany sequence within the duplicated region of the rhg1 locus.

Methods are also provided for identifying and/or selecting a soybeanplant or a soybean germplasm with enhanced resistance to cyst nematodeby detecting an increased copy number of at least one of or anycombination of Glyma18g2580 (SEQ ID NO: 3), Glyma18g2590 (SEQ ID NO: 2),Glyma18g2600 (SEQ ID NO:4), Glyma18g2610 (SEQ ID NO:5); and Glyma18g2570(SEQ ID NO:1) or an active variant or fragment thereof.

Methods of identifying and/or selecting a soybean plant or a soybeangermplasm with enhanced resistance to soybean cyst nematode are alsoprovided which comprise detecting the DNA junction formed at thebreakpoint of a duplication of nucleotide sequences within the rhg1locus.

Kits for the various methods of identifying the soybean plants orsoybean germplasm having the enhanced resistance to SCN are furtherprovided.

Transgenic plants, plant parts, and seed comprising a heterologouspolynucleotide operably linked to a promoter active in the plant areprovided, as are methods of making such plants and methods of use,wherein said heterologous polynucleotide comprises at least one, or anycombination thereof, of Glyma18g2580 (SEQ ID NO: 3), Glyma18g2590 (SEQID NO: 2), Glyma18g2600 (SEQ ID NO:4), Glyma18g2610 (SEQ ID NO:5); andGlyma18g2570 (SEQ ID NO:1) or an active variant or fragment thereof.Expression of the heterologous polynucleotide enhances the resistance ofthe plant to cyst nematode.

DETAILED DESCRIPTION OF DRAWINGS

FIG. 1 shows the heterozygosity within P188788 derived rhg1 locus.Haplotypes were built from SNPs found during deep resequencing of Soyancestors. Persistent heterozygosity inherited over generations at therhg1 locus within resistant lines derived from PI88788 was observed.

FIG. 2 shows the next-generation sequencing alignments that indicatetandem duplication. Read alignments are visualized as grey boxes intracks with read-pairs connected by grey lines. Total sequencingcoverage is visualized in the track above with polymorphic sites.Significant changes in coverage are observed at the putative duplicationbreakpoints (arrows). Discordantly mapped paired-end reads (circles)suggest tandem duplication of the interval Gm18:1.632-1.663 (Mb).

FIG. 3 shows a copy-number analysis and indicates a substantialcopy-number increase. Copy-number analysis was performed by calculatingthe sequencing coverage in a moving window across the rhg1 fine-mapregion. Coverage of Peking indicated an increased normalized coverageratio in the region of Gm18:1.632-1.663 (Mb) of approximately 3-foldover susceptible. Coverage of PI88788 indicated an increased normalizedcoverage ratio of 9-fold in the same region.

FIG. 4 shows the results of qPCR analysis which indicates an increasedcopy-number in Peking and PI88788. PCR primers were designed against thesingle-copy and variable-copy regions of the rhg1 fine-mapped locus. Twosources of Peking and PI88788 resistance and three susceptible lineswere assayed in two replicates for all primer-pairs. Replicates wereaveraged and ΔΔCt analysis was performed pair-wise and averaged betweenevery variable-copy and single-copy combination. The results areconsistent with an ˜4-fold and ˜9-fold increased copy-number in Pekingand PI88788 relative to susceptible lines.

FIG. 5 shows the Blast results of a contig spanning the duplicationbreakpoints. Discordantly mapped paired-end reads from the boundaries ofthe tandem duplication were assembled into contigs. Contigs were blastedagainst the Soybean Genomic Assembly Glyma1.01 (JGI). The alignment ofthis contig to the reference is consistent with a tandem duplicationevent with breakpoints at Gm18:1663448 and Gm18:1632228. Query A is setforth in SEQ ID NO: 18; Subject A is set forth in SEQ ID NO: 19; Query Bis set forth in SEQ ID NO: 20; and subject B is set forth in SEQ IDNO:21.

FIG. 6 shows PCR amplicons in Peking and PI88788 support the putativebreakpoints. PCR primer-pairs were designed to amplify the ends of thecopy-variable regions (dark (or black) pair, light (or gray) pair) andacross the putative breakpoints (mixed pair). Primer-pairs designed toamplify the ends of the copy-variable region produce amplicons in alllines. Primer-pairs designed to amplify across the putative breakpointsproduce amplicons in Peking and PI88788 but not susceptible.

FIG. 7 shows sequencing and alignment of PCR amplicons confirms thebreakpoints. A primer pair spanning the breakpoint junction was orderedwith the M13 sequencing tags, tested and sent off for Sanger Sequencing.The resulting amplicons from Peking and PI88788 were aligned to theSoybean Genomic Assembly Glyma1.01 (JGI) by blast. The alignment ofthese amplicons to the reference is consistent with a tandem duplicationevent with breakpoints at Gm18:1663448 and Gm18:1632228, an example isdepicted. Query A is set forth in SEQ ID NO: 22; subject A is set forthin SEQ ID NO: 23; query B is set forth in SEQ ID NO:24; and subject B isset forth in SEQ ID NO: 25.

FIG. 8 provides various primer sequences.

DETAILED DESCRIPTION I. Definitions

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particular embodiments,which can, of course, vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to be limiting.

Certain definitions used in the specification and claims are providedbelow. In order to provide a clear and consistent understanding of thespecification and claims, including the scope to be given such terms,the following definitions are provided.

As used in this specification and the appended claims, terms in thesingular and the singular forms “a”, “an” and “the”, for example,include plural referents unless the content clearly dictates otherwise.Thus, for example, reference to “plant”, “the plant” or “a plant” alsoincludes a plurality of plants; also, depending on the context, use ofthe term “plant” can also include genetically similar or identicalprogeny of that plant; use of the term “a nucleic acid” optionallyincludes, as a practical matter, many copies of that nucleic acidmolecule; similarly, the term “probe” optionally (and typically)encompasses many similar or identical probe molecules.

Additionally, as used herein, “comprising” is to be interpreted asspecifying the presence of the stated features, integers, steps, orcomponents as referred to, but does not preclude the presence oraddition of one or more features, integers, steps, or components, orgroups thereof. Thus, for example, a kit comprising one pair ofoligonucleotide primers may have two or more pairs of oligonucleotideprimers. Additionally, the term “comprising” is intended to includeexamples encompassed by the terms “consisting essentially of” and“consisting of.” Similarly, the term “consisting essentially of” isintended to include examples encompassed by the term “consisting of.”

“Allele” means any of one or more alternative forms of a geneticsequence. In a diploid cell or organism, the two alleles of a givensequence typically occupy corresponding loci on a pair of homologouschromosomes. With regard to a SNP marker, allele refers to the specificnucleotide base present at that SNP locus in that individual plant.

The term “amplifying” in the context of nucleic acid amplification isany process whereby additional copies of a selected nucleic acid (or atranscribed form thereof) are produced. An “amplicon” is an amplifiednucleic acid, e.g., a nucleic acid that is produced by amplifying atemplate nucleic acid by any available amplification method.

“Backcrossing” is a process in which a breeder crosses a progeny varietyback to one of the parental genotypes one or more times.

The term “chromosome segment” designates a contiguous linear span ofgenomic DNA that resides in planta on a single chromosome. “Chromosomeinterval” refers to a chromosome segment defined by specific flankingmarker loci.

“Cultivar” and “variety” are used synonymously and mean a group ofplants within a species (e.g., Glycine max) that share certain genetictraits that separate them from other possible varieties within thatspecies. Soybean cultivars are inbred lines produced after severalgenerations of self-pollinations. Individuals within a soybean cultivarare homogeneous, nearly genetically identical, with most loci in thehomozygous state.

An “elite line” is an agronomically superior line that has resulted frommany cycles of breeding and selection for superior agronomicperformance. Numerous elite lines are available and known to those ofskill in the art of soybean breeding.

An “elite population” is an assortment of elite individuals or linesthat can be used to represent the state of the art in terms ofagronomically superior genotypes of a given crop species, such assoybean.

An “exotic soybean strain” or an “exotic soybean germplasm” is a strainor germplasm derived from a soybean not belonging to an available elitesoybean line or strain of germplasm. In the context of a cross betweentwo soybean plants or strains of germplasm, an exotic germplasm is notclosely related by descent to the elite germplasm with which it iscrossed. Most commonly, the exotic germplasm is not derived from anyknown elite line of soybean, but rather is selected to introduce novelgenetic elements (typically novel alleles) into a breeding program.

A “genetic map” is a description of genetic linkage relationships amongloci on one or more chromosomes (or linkage groups) within a givenspecies, generally depicted in a diagrammatic or tabular form.

“Genotype” refers to the genetic constitution of a cell or organism.

“Germplasm” means the genetic material that comprises the physicalfoundation of the hereditary qualities of an organism. As used herein,germplasm includes seeds and living tissue from which new plants may begrown; or, another plant part, such as leaf, stem, pollen, or cells,that may be cultured into a whole plant. Germplasm resources providesources of genetic traits used by plant breeders to improve commercialcultivars.

An individual is “homozygous” if the individual has only one type ofallele at a given locus (e.g., a diploid individual has a copy of thesame allele at a locus for each of two homologous chromosomes). Anindividual is “heterozygous” if more than one allele type is present ata given locus (e.g., a diploid individual with one copy each of twodifferent alleles). The term “homogeneity” indicates that members of agroup have the same genotype at one or more specific loci. In contrast,the term “heterogeneity” is used to indicate that individuals within thegroup differ in genotype at one or more specific loci.

“Introgression” means the entry or introduction of a gene, QTL, marker,haplotype, marker profile, trait, or trait locus from the genome of oneplant into the genome of another plant.

The terms “label” and “detectable label” refer to a molecule capable ofdetection. A detectable label can also include a combination of areporter and a quencher, such as are employed in FRET probes or TaqMan™probes. The term “reporter” refers to a substance or a portion thereofwhich is capable of exhibiting a detectable signal, which signal can besuppressed by a quencher. The detectable signal of the reporter is,e.g., fluorescence in the detectable range. The term “quencher” refersto a substance or portion thereof which is capable of suppressing,reducing, inhibiting, etc., the detectable signal produced by thereporter. As used herein, the terms “quenching” and “fluorescence energytransfer” refer to the process whereby, when a reporter and a quencherare in close proximity, and the reporter is excited by an energy source,a substantial portion of the energy of the excited state non-radiativelytransfers to the quencher where it either dissipates non-radiatively oris emitted at a different emission wavelength than that of the reporter.

A “line” or “strain” is a group of individuals of identical parentagethat are generally inbred to some degree and that are generallyhomozygous and homogeneous at most loci (isogenic or near isogenic). A“subline” refers to an inbred subset of descendents that are geneticallydistinct from other similarly inbred subsets descended from the sameprogenitor. Traditionally, a subline has been derived by inbreeding theseed from an individual soybean plant selected at the F3 to F5generation until the residual segregating loci are “fixed” or homozygousacross most or all loci. Commercial soybean varieties (or lines) aretypically produced by aggregating (“bulking”) the self-pollinatedprogeny of a single F3 to F5 plant from a controlled cross between 2genetically different parents. While the variety typically appearsuniform, the self-pollinating variety derived from the selected planteventually (e.g., F8) becomes a mixture of homozygous plants that canvary in genotype at any locus that was heterozygous in the originallyselected F3 to F5 plant. Marker-based sublines that differ from eachother based on qualitative polymorphism at the DNA level at one or morespecific marker loci are derived by genotyping a sample of seed derivedfrom individual self-pollinated progeny derived from a selected F3-F5plant. The seed sample can be genotyped directly as seed, or as planttissue grown from such a seed sample. Optionally, seed sharing a commongenotype at the specified locus (or loci) are bulked providing a sublinethat is genetically homogenous at identified loci important for a traitof interest (e.g., yield, tolerance, etc.).

“Linkage” refers to a phenomenon wherein alleles on the same chromosometend to segregate together more often than expected by chance if theirtransmission was independent. Genetic recombination occurs with anassumed random frequency over the entire genome. Genetic maps areconstructed by measuring the frequency of recombination between pairs oftraits or markers. The closer the traits or markers are to each other onthe chromosome, the lower the frequency of recombination, and thegreater the degree of linkage. Traits or markers are considered hereinto be linked if they generally co-segregate. A 1/100 probability ofrecombination per generation is defined as a map distance of 1.0centiMorgan (1.0 cM). The genetic elements or genes located on a singlechromosome segment are physically linked. Two loci can be located inclose proximity such that recombination between homologous chromosomepairs does not occur between the two loci during meiosis with highfrequency, e.g., such that linked loci co-segregate at least about 90%of the time, e.g., 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%,99.75%, or more of the time. The genetic elements located within achromosome segment are also genetically linked, typically within agenetic recombination distance of less than or equal to 50 centimorgans(cM), e.g., about 49, 40, 30, 20, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75,0.5, or 0.25 cM or less. That is, two genetic elements within a singlechromosome segment undergo recombination during meiosis with each otherat a frequency of less than or equal to about 50%, e.g., about 49%, 40%,30%, 20%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, or 0.25%or less. Closely linked markers display a cross over frequency with agiven marker of about 10% or less (the given marker is within about 10cM of a closely linked marker). Put another way, closely linked locico-segregate at least about 90% of the time. With regard to physicalposition on a chromosome, closely linked markers can be separated, forexample, by about 1 megabase (Mb; 1 million nucleotides), about 500kilobases (Kb; 1000 nucleotides), about 400 Kb, about 300 Kb, about 200Kb, about 100 Kb, about 50 Kb, about 25 Kb, about 10 Kb, about 5 Kb,about 4 Kb, about 3 Kb, about 2 Kb, about 1 Kb, about 500 nucleotides,about 250 nucleotides, or less.

When referring to the relationship between two genetic elements, such asa genetic element contributing to tolerance and a proximal marker,“coupling” phase linkage indicates the state where the “favorable”allele at the tolerance locus is physically associated on the samechromosome strand as the “favorable” allele of the respective linkedmarker locus. In coupling phase, both favorable alleles are inheritedtogether by progeny that inherit that chromosome strand. In “repulsion”phase linkage, the “favorable” allele at the locus of interest (e.g., aQTL for tolerance) is physically linked with an “unfavorable” allele atthe proximal marker locus, and the two “favorable” alleles are notinherited together (i.e., the two loci are “out of phase” with eachother).

“Linkage disequilibrium” refers to a phenomenon wherein alleles tend toremain together in linkage groups when segregating from parents tooffspring, with a greater frequency than expected from their individualfrequencies.

“Linkage group” refers to traits or markers that generally co-segregate.A linkage group generally corresponds to a chromosomal region containinggenetic material that encodes the traits or markers.

“Locus” is a defined segment of DNA.

A “map location” or “map position” or “relative map position” is anassigned location on a genetic map relative to linked genetic markerswhere a specified marker can be found within a given species. Mappositions are generally provided in centimorgans. A “physical position”or “physical location” or “physical map location” is the position,typically in nucleotide bases, of a particular nucleotide, such as a SNPnucleotide, on a chromosome.

“Mapping” is the process of defining the linkage relationships of locithrough the use of genetic markers, populations segregating for themarkers, and standard genetic principles of recombination frequency.

“Marker” or “molecular marker” is a term used to denote a nucleic acidor amino acid sequence that is sufficiently unique to characterize aspecific locus on the genome. Any detectible polymorphic trait can beused as a marker so long as it is inherited differentially and exhibitslinkage disequilibrium with a phenotypic trait of interest. A number ofsoybean markers have been mapped and linkage groups created, asdescribed in Cregan, P. B., et al., “An Integrated Genetic Linkage Mapof the Soybean Genome” (1999) Crop Science 39:1464-90, and more recentlyin Choi, et al., “A Soybean Transcript Map: Gene Distribution, Haplotypeand Single-Nucleotide Polymorphism Analysis” (2007) Genetics 176:685-96.Many soybean markers are publicly available at the USDA affiliatedsoybase website (www.soybase.org). All markers are used to define aspecific locus on the soybean genome. Large numbers of these markershave been mapped. Each marker is therefore an indicator of a specificsegment of DNA, having a unique nucleotide sequence. The map positionsprovide a measure of the relative positions of particular markers withrespect to one another. When a trait is stated to be linked to a givenmarker it will be understood that the actual DNA segment whose sequenceaffects the trait generally co-segregates with the marker. More preciseand definite localization of a trait can be obtained if markers areidentified on both sides of the trait. By measuring the appearance ofthe marker(s) in progeny of crosses, the existence of the trait can bedetected by relatively simple molecular tests without actuallyevaluating the appearance of the trait itself, which can be difficultand time-consuming because the actual evaluation of the trait requiresgrowing plants to a stage and/or under environmental conditions wherethe trait can be expressed. Molecular markers have been widely used todetermine genetic composition in soybeans. “Marker assisted selection”refers to the process of selecting a desired trait or traits in a plantor plants by detecting one or more nucleic acids from the plant, wherethe nucleic acid is linked to the desired trait, and then selecting theplant or germplasm possessing those one or more nucleic acids.

“Haplotype” refers to a combination of particular alleles present withina particular plant's genome at two or more linked marker loci, forinstance at two or more loci on a particular linkage group. Forinstance, in one example, two specific marker loci on LG-O are used todefine a haplotype for a particular plant. In still further examples, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or morelinked marker loci are used to define a haplotype for a particularplant.

The term “plant” includes reference to an immature or mature wholeplant, including a plant from which seed or grain or anthers have beenremoved. Seed or embryo that will produce the plant is also consideredto be the plant.

“Plant parts” means any portion or piece of a plant, including leaves,stems, buds, roots, root tips, anthers, seed, grain, embryo, pollen,ovules, flowers, cotyledons, hypocotyls, pods, flowers, shoots, stalks,tissues, tissue cultures, cells, and the like. Grain is intended to meanthe mature seed produced by commercial growers for purposes other thangrowing or reproducing the species.

“Polymorphism” means a change or difference between two related nucleicacids. A “nucleotide polymorphism” refers to a nucleotide that isdifferent in one sequence when compared to a related sequence when thetwo nucleic acids are aligned for maximal correspondence.

“Polynucleotide,” “polynucleotide sequence,” “nucleic acid sequence,”“nucleic acid fragment,” and “oligonucleotide” are used interchangeablyherein. These terms encompass nucleotide sequences and the like. Apolynucleotide is a polymer of nucleotides that is single- ormulti-stranded, that optionally contains synthetic, non-natural, oraltered RNA or DNA nucleotide bases. A DNA polynucleotide may becomprised of one or more strands of cDNA, genomic DNA, synthetic DNA, ormixtures thereof.

“Primer” refers to an oligonucleotide (synthetic or occurringnaturally), which is capable of acting as a point of initiation ofnucleic acid synthesis or replication along a complementary strand whenplaced under conditions in which synthesis of a complementary strand iscatalyzed by a polymerase. Typically, primers are about 10 to 30nucleotides in length, but longer or shorter sequences can be employed.Primers may be provided in double-stranded form, though thesingle-stranded form is more typically used. A primer can furthercontain a detectable label, for example a 5′ end label.

“Probe” refers to an oligonucleotide (synthetic or occurring naturally)that is complementary (though not necessarily fully complementary) to apolynucleotide of interest and forms a duplexed structure byhybridization with at least one strand of the polynucleotide ofinterest. Typically, probes are oligonucleotides from 10 to 50nucleotides in length, but longer or shorter sequences can be employed.A probe can further contain a detectable label.

“Quantitative trait loci” or “QTL” refer to the genetic elementscontrolling a quantitative trait.

“Recombination frequency” is the frequency of a crossing over event(recombination) between two genetic loci. Recombination frequency can beobserved by following the segregation of markers and/or traits duringmeiosis.

“Tolerance and “improved tolerance” are used interchangeably herein andrefer to any type of increase in resistance or tolerance to, or any typeof decrease in susceptibility. A “tolerant plant” or “tolerant plantvariety” need not possess absolute or complete tolerance. Instead, a“tolerant plant,” “tolerant plant variety,” or a plant or plant varietywith “improved tolerance” will have a level of resistance or tolerancewhich is higher than that of a comparable susceptible plant or variety.

“Self crossing” or “self pollination” or “selfing” is a process throughwhich a breeder crosses a plant with itself; for example, a secondgeneration hybrid F2 with itself to yield progeny designated F2:3.

“SNP” or “single nucleotide polymorphism” means a sequence variationthat occurs when a single nucleotide (A, T, C, or G) in the genomesequence is altered or variable. “SNP markers” exist when SNPs aremapped to sites on the soybean genome.

The term “yield” refers to the productivity per unit area of aparticular plant product of commercial value. For example, yield ofsoybean is commonly measured in bushels of seed per acre or metric tonsof seed per hectare per season. Yield is affected by both genetic andenvironmental factors. Yield is the final culmination of all agronomictraits.

A “subject plant or plant cell” is one in which genetic alteration, suchas transformation, has been affected as to a gene of interest, or is aplant or plant cell which is descended from a plant or cell so alteredand which comprises the alteration. A “control” or “control plant” or“control plant cell” provides a reference point for measuring changes inphenotype of the subject plant or plant cell.

A control plant or plant cell may comprise, for example: (a) a wild-typeplant or cell, i.e., of the same genotype as the starting material forthe genetic alteration which resulted in the subject plant or cell; (b)a plant or plant cell of the same genotype as the starting material butwhich has been transformed with a null construct (i.e. with a constructwhich has no known effect on the trait of interest, such as a constructcomprising a marker gene); (c) a plant or plant cell which is anon-transformed segregant among progeny of a subject plant or plantcell; (d) a plant or plant cell genetically identical to the subjectplant or plant cell but which is not exposed to conditions or stimulithat would induce expression of the gene of interest; or (e) the subjectplant or plant cell itself, under conditions in which the gene ofinterest is not expressed.

As used herein, an “isolated” or “purified” polynucleotide orpolypeptide, or biologically active portion thereof, is substantially oressentially free from components that normally accompany or interactwith the polynucleotide or polypeptide as found in its naturallyoccurring environment. Thus, an isolated or purified polynucleotide orpolypeptide is substantially free of other cellular material or culturemedium when produced by recombinant techniques, or substantially free ofchemical precursors or other chemicals when chemically synthesized.Optimally, an “isolated” polynucleotide is free of sequences (optimallyprotein encoding sequences) that naturally flank the polynucleotide(i.e., sequences located at the 5′ and 3′ ends of the polynucleotide) inthe genomic DNA of the organism from which the polynucleotide isderived. For example, in various embodiments, the isolatedpolynucleotide can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb,0.5 kb, or 0.1 kb of nucleotide sequence that naturally flank thepolynucleotide in genomic DNA of the cell from which the polynucleotideis derived. A polypeptide that is substantially free of cellularmaterial includes preparations of polypeptides having less than about30%, 20%, 10%, 5%, or 1% (by dry weight) of contaminating protein. Whenthe polypeptide of the invention or biologically active portion thereofis recombinantly produced, optimally culture medium represents less thanabout 30%, 20%, 10%, 5%, or 1% (by dry weight) of chemical precursors ornon-protein-of-interest chemicals.

Standard recombinant DNA and molecular cloning techniques used hereinare well known in the art and are described more fully in Sambrook, J.,Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual;Cold Spring Harbor Laboratory Press Cold Spring Harbor, 1989(hereinafter “Sambrook”).

II. Overview

Plants that have enhanced resistance to soybean cyst nematode (SCN) areknown, however the reason for their resistance has been unknown untilnow. The present invention shows that duplications within the rhg1locus, and subsequent increase in expression of the duplicated geneswithin the duplicated segment, causes the resistance within the soybeanto this economically important soybean disease. More specifically, atandem duplication within the rhg1 fine-mapped region has beenidentified and evidence is provided to suggest that rhg1 and rhg1-bharbor copy-number variable alleles of this structural variant. Thiscopy-variation underlies the SCN resistance phenotype attributed to therhg1 locus. These results have important implications for soybeanproduct development and point to potential strategies for cultivarimprovement. The various methods and compositions provided herein applythis new understanding of the rhg1- and rhg1-b alleles.

By “enhanced resistance” is intended that the plants show a decrease inthe disease symptoms that are the outcome of plant-cyst nematodeinteractions. That is, the damage caused by cyst nematode is prevented,or alternatively, the disease symptoms caused by the cyst nematode isminimized or lessened. Thus, enhanced resistance to cyst nematode canresult in the suppressing, controlling, and/or killing the invading cystnematode. In specific embodiments, the enhanced resistance can reducethe disease symptoms resulting from pathogen challenge by at least about2% to at least about 6%, at least about 5% to about 50%, at least about10% to about 60%, at least about 30% to about 70%, at least about 40% toabout 80%, or at least about 50% to about 90% or greater. Hence, themethods provided herein can be utilized to protect plants from disease,particularly those diseases that are caused by cyst nematodes. Assaysthat measure the control of a pest are known and include measuring overtime, the average lesion diameter, the pathogen biomass, and the overallpercentage of decayed plant tissues. See, for example, Thomma et al.(1998) Plant Biology 95:15107-15111 and U.S. Pat. No. 5,614,395, both ofwhich are herein incorporated by reference.

A variety of cyst nematodes are known. Particular members of the cystnematodes, include, but are not limited to, Heterodera glycines (soybeancyst nematode); Heterodera schachtii (beet cyst nematode); Heteroderaavenae (cereal cyst nematode); and Globodera rostochiensis and Globoderapailida (potato cyst nematodes). In specific embodiments, the methodsand compositions disclosed herein are employed to enhance resistance toHeterodera glycines (soybean cyst nematode).

III. Region Conferring Enhanced Resistance to Cyst Nematodes

As used herein, the rhg1 locus comprises a region of the soybean genomewhich maps to linkage group G. See, for example, Kim et al. (2010) ThePlant Genome Journal 32:81-89 which is herein incorporated by referencein its entirety. Two rhg1 alleles that confer resistance to a cystnematode are known and include the rhg1 allele derived from Peking andthe rhg1-b allele derived from PI88788. As described elsewhere herein,characterization of the rhg1 alleles conferring resistance to the cystnematode comprises an increase in copy number of at least one region ofthe rhg1 locus.

In specific embodiments, an increase in copy number comprises aduplication of at least one region within the rhg1 locus. A“duplication” of at least one region within the rhg1 locus can compriseany increase in copy number of that region including 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15 or more copies. A region within the rhg1locus can be of any length, including 20, 100, 200, 300, 400nucleotides, 0.5, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 40, 50,60 KB or longer.

In specific embodiments, the duplicated region of the rhg1 locus isbetween about position GM18:1663448 and about position GM18:1632228 ofthe soybean genome. In still further embodiments, the duplication of theat least one region of the rhg1 locus comprises a tandem duplication ofthe region. As used herein, the term “tandem” refers to sequences beingimmediately adjacent to one another.

In one embodiment, the duplication of the region within the rhg1 locuscomprises a tandem duplication of the soybean genome between aboutposition GM18:1663448 and about position GM18:1632228. In furtherembodiments, the tandem duplications are found in the same orientationwith respect to one another.

In other embodiments, the duplication of the region within the rhg1locus comprises a duplication of at least one gene or regulatory regionwithin the locus or it can comprise a duplication of at least one geneor regulatory region located between about position GM18:1663448 andabout position GM18:1632228 of the soybean genome. The genes withinthese regions can encode polypeptides or RNA regulatory elements. Inspecific embodiments, the duplication of the region within the rhg1locus comprises an increase in copy of number of anyone or anycombination of the following polynucleotides: Glyma18g2580 (SEQ ID NO:3), Glyma18g2590 (SEQ ID NO: 2), Glyma18g2600 (SEQ ID NO:4),Glyma18g2610 (SEQ ID NO:5); and/or Glyma18g2570 (SEQ ID NO:1) or anactive variant or fragment thereof.

IV. Methods of Identifying and Breeding Plants Having an EnhancedResistance to Cyst Nematodes

Various methods are provided to identify soybean plants with an enhancedresistance to cyst nematodes. In one embodiment, the method ofidentifying comprises detecting at least one marker allele associatedwith a duplication of at least one region within the rhg1 locus. Theterm “associated with” in connection with a relationship between amarker locus and a phenotype refers to a statistically significantdependence of marker frequency with respect to a quantitative scale orqualitative gradation of the phenotype. Thus, an allele of a marker isassociated with a trait of interest when the allele of the marker locusand the trait phenotypes are found together in the progeny of anorganism more often than if the marker genotypes and trait phenotypessegregated separately.

In one embodiment, the marker allele being detected is associated with(1) a duplication or a tandem duplication of the soybean genome betweenabout position GM18:1663448 and about position GM18:1632228; (2) aduplicated region found between about position GM18:1663448 and aboutposition GM18:1632228; or (4) a duplication of a gene between foundbetween about position GM18:1663448 and about position GM18:1632228;wherein each of the duplications is associated with an enhancedresistance to cyst nematode.

In one non-limiting example, the marker allele associated with theduplication of at least one region within the rhg1 locus comprises theDNA junction formed at the breakpoint of a tandem duplication of aregion within the rhg1 locus. Such DNA junction regions are described inmore detail elsewhere herein.

Additional markers alleles that can be used are set forth in Tables 1and 2. Table 1 provides a list of polymorphic sites found within therhg1 locus. The chromosomal position is denoted in the first twocolumns. “Ref” denotes the nucleotide occurring in the reference soybeansample, and “ALT” denotes the nucleotide found in the soybean lineshaving enhanced resistance to SCN. The presence of the nucleotidealteration in the Peking and the P188788 lines is denoted in the lasttwo columns. Table 2 provides a list of polymorphic sites found withinthe coding regions of the rhg1 locus, specifically polymorphisms inGlyma18g2580 (SEQ ID NO: 3), Glyma18g2590 (SEQ ID NO: 2), Glyma18g2600(SEQ ID NO:4), Glyma18g2610 (SEQ ID NO:5); and Glyma18g2570 (SEQ IDNO:1) are provided. Thus, any one marker or any combination of thepolymorphisms set forth in Tables 1 and 2 can be used to aid inidentifying and selecting soybean plants with enhanced resistance toSCN.

Further provided are methods for identifying a soybean plant or asoybean germplasm with enhanced resistance to cyst nematode. The methodcomprises detecting a duplication of a region within the rhg1 locuswithin the genome of the soybean plant or germplasm. In such a method,the duplication of a region within the rhg1 locus can comprise a tandemduplication of the region of the soybean genome between about positionGM18:1663448 and about position GM18:1632228. In other embodiments, theduplication of the region within the rhg1 locus comprises a duplicationof any region between position GM18:1663448 and GM18:1632228, including,for example, at least one of or any combination of Glyma18g2580 (SEQ IDNO: 3), Glyma18g2590 (SEQ ID NO: 2), Glyma18g2600 (SEQ ID NO:4),Glyma18g2610 (SEQ ID NO:5); and Glyma18g2570 (SEQ ID NO:1) or activevariants and fragments thereof. Such methods of detection includequantitative PCR or other quantitative techniques, which are describedin further detail elsewhere herein.

Additional methods for identifying a soybean plant or a soybeangermplasm with enhanced resistance to cyst nematode include detecting anincreased copy number of any duplicated region within the rhg1 locus, orbetween genomic position GM18:1663448 and GM18:1632228, or detecting anincreased copy number of any one or any combination of the followingpolynucleotides Glyma18g2580 (SEQ ID NO: 3), Glyma18g2590 (SEQ ID NO:2), Glyma18g2600 (SEQ ID NO:4), Glyma18g2610 (SEQ ID NO:5); andGlyma18g2570 (SEQ ID NO:1), or an active variant or fragment thereof.Methods by which copy number can be assayed are described elsewhereherein, and include, for example, PCR amplification and DNA sequence orthe copy number analysis as shown in the examples herein. An increase incopy number includes at least 2, 3, 4, 5, 6, 7, 8, 9, 10 or more copiesof a given region.

Further provided are methods for identifying a soybean plant or asoybean germplasm with enhanced resistance to cyst nematodes bydetecting the DNA junction formed at the breakpoint of a duplication ofa region within the rhg1 locus. A “junction” is a point where twospecific DNA fragments join. As used herein, a “DNA junction” refers toDNA that comprises a junction point. For example, a junction existswhere the duplicated region of the rhg1 locus joins the flanking genomicDNA. Thus, as used herein, a “DNA junction formed at the breakpoint of aduplication of a region” comprises the nucleotide sequence appearing atthe junction where the two regions of DNA are repeated. In specificembodiments, the duplications are repeated in tandem. In one embodiment,the DNA junction comprises a DNA sequence that arises when the region ofthe soybean genome between about position GM18:1663448 and aboutposition GM18:1632228 are placed in tandem and in the same orientationwith itself. The DNA junction can be of any length including, but notlimited to, 10 nt, 20 nt, 30 nt, 40 nt, 50 nt, 75 nt, 100 nt, 150 nt,200 nt, 250 nt, 300 nt, 400 nt, 500 nt, 600 nt, 700 nt or more. Thelength of the DNA junction should be of sufficient length to allow forthe detection of the junction and, depending on the need and detectiontechnique being employed, to allow for a sufficient level of specificityof detection.

In specific embodiments, the DNA junction formed at the breakpoint ofthe tandem duplication of a region within the rhg1 locus comprises thesequence set forth in any one of SEQ ID NOS: 6, 7, 8 or 9 or a fragmentthereof. In specific embodiments, the DNA junction being detectedcomprises a fragment of any one of SEQ ID NO: 6, 7, 8 or 9 having atleast 10 nt, 20 nt, 30 nt, 40 nt, 50 nt, 75 nt, 100 nt, 150 nt, 200 nt,250 nt, 300 nt or more consecutive nucleotides in length.

Various methods can be used to detect the novel DNA junction including,but not limited to, PCR amplification, hybridization methods or DNAsequencing. Such methods are discussed elsewhere herein.

In one embodiment, detecting a DNA junction comprises contacting a plantmaterial with a first and a second primer; and, amplifying apolynucleotide comprising a DNA junction formed at the breakpoint of aduplication of a region of the rhg1 locus. In more specific embodiments,the first and second primer amplify a polynucleotide comprising a DNAjunction comprising a DNA sequence that arises when the region of thesoybean genome between about position GM18:1663448 and about positionGM18:1632228 are placed in tandem and in the same orientation withitself. In specific embodiments, the primer pair amplifies the DNAjunction set forth in any one of SEQ ID NOS: 6, 7, 8, or 9 or a fragmentthereof.

As used herein, “plant material” refers to material which is obtained orderived from a plant or plant part. In specific embodiments, thebiological sample comprises a soybean tissue.

The polynucleotide probes and primers employed in the various methodsand kits disclosed herein specifically detect a target DNA sequence. Anyconventional nucleic acid hybridization or amplification method can beused to identify the presence of the desired junction DNA. By“specifically detect” is intended that the polynucleotide can be usedeither as a primer to amplify the desired DNA sequence or thepolynucleotide can be used as a probe that hybridizes under stringentconditions to a polynucleotide having the desired DNA sequence. Thelevel or degree of hybridization which allows for the specific detectionof the desired DNA sequence is sufficient to distinguish thepolynucleotide with the desired DNA sequence from a polynucleotidelacking this region and thereby allow for discriminately identifying aplant having the desired DNA sequence.

By “shares sufficient sequence identity or complentarity to allow forthe amplification of a desired DNA sequence” is intended the sequenceshares at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,99% or 100% identity or complementarity to a fragment or across the fulllength of the polynucleotide having the desired DNA sequence.

Regarding the amplification of a target polynucleotide (e.g., by PCR)using a particular amplification primer pair, “stringent conditions” areconditions that permit the primer pair to hybridize to the targetpolynucleotide to which a primer having the corresponding wild-typesequence (or its complement) would bind and preferably to produce anidentifiable amplification product (the amplicon) having a (1) a DNAjunction comprising the breakpoint of a duplication of a region withinthe rhg1 locus; (2) DNA junction comprising a DNA sequence that ariseswhen the region of the soybean genome between about positionGM18:1663448 and about position GM18:1632228 are placed in tandem and inthe same orientation with itself; (3) a DNA junction set forth in anyone of SEQ ID NOS: 6, 7, 8, or 9 or a fragment thereof; or (4) any DNAsequence that is associated with a duplication within the rhg1 locus.Ina PCR approach, oligonucleotide primers can be designed for use in PCRreactions to amplify the desired DNA junction. Methods for designing PCRprimers and PCR cloning are generally known in the art and are disclosedin Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual (2ded., Cold Spring Harbor Laboratory Press, Plainview, N.Y.). See alsoInnis et al., eds. (1990) PCR Protocols: A Guide to Methods andApplications (Academic Press, New York); Innis and Gelfand, eds. (1995)PCR Strategies (Academic Press, New York); and Innis and Gelfand, eds.(1999) PCR Methods Manual (Academic Press, New York). Methods ofamplification are further described in U.S. Pat. Nos. 4,683,195,4,683,202 and Chen et al. (1994) PNAS 91:5695-5699. These methods aswell as other methods known in the art of DNA amplification may be usedin the practice of the embodiments of the present invention. It isunderstood that a number of parameters in a specific PCR protocol mayneed to be adjusted to specific laboratory conditions and may beslightly modified and yet allow for the collection of similar results.

The amplified polynucleotide (amplicon) can be of any length. Forexample, the amplicon can be about 10, 50, 100, 200, 300, 500, 700, 100,2000, 3000, 4000, 5000 nucleotides in length or longer.

Any primer can be employed in the methods of the invention that allows a(1) a DNA junction comprising the breakpoint of a duplication of aregion within the rhg1 locus; (2) a DNA junction comprising a DNAsequence that arises when the region of the soybean genome between aboutposition GM18:1663448 and about position GM18:1632228 are placed intandem and in the same orientation with itself; (3) a DNA junction setforth in any one of SEQ ID NOS: 6, 7, 8, or 9 or a fragment thereof; or(4) any DNA sequence that is associated with a duplication of the rhg1locus to be amplified and/or detected. For example, in specificembodiments, the first primer comprises a fragment of a polynucleotidesequence flanking the 3′ end of the DNA junction and the second primercomprises a fragment of a polynucleotide sequence flanking the 5′end ofthe DNA junction, wherein the first or the second primer sharessufficient sequence identity or complementarity to the polynucleotide toamplify desired DNA junction region. The primers can be of any lengthsufficient to amplify the desired region including, for example, atleast 6, 7, 8, 9, 10, 15, 20, 15, or or about 7-10, 10-15, 15-20, 20-25,25-30, 30-35, 35-40, 40-45 nucleotides or longer. In one embodiment, theprimer pair employed to amplify the junction comprises SEQ ID NO: 14 and15. Kits having the primer pair to allow for the amplification of thedesired junction are further provided.

Thus, in specific embodiments, a method of identifying a plant withenhanced resistance to cyst nematode comprising detecting the DNAjunction formed at the breakpoint of a duplicated region within the rhg1locus is provided. The method comprises (a) extracting a DNA sample fromthe soybean plant; (b) providing a pair of DNA primer molecules that canspecifically amplify the desired DNA junction, (c) providing DNAamplification reaction conditions; (d) performing the DNA amplificationreaction, thereby producing a DNA amplicon molecule; and (e) detectingthe DNA amplicon molecule, wherein the detection of said DNA ampliconmolecule in the DNA amplification reaction indicates the presence of asoybean plant having enhanced resistance to cyst nematodes. In order fora nucleic acid molecule to serve as a primer or probe it need only besufficiently complementary in sequence to be able to form a stabledouble-stranded structure under the particular solvent and saltconcentrations employed.

In hybridization techniques, all or part of a polynucleotide thatselectively hybridizes to a target polynucleotide having the desired DNAjunction is employed. By “stringent conditions” or “stringenthybridization conditions” when referring to a polynucleotide probe isintended conditions under which a probe will hybridize to its targetsequence to a detectably greater degree than to other sequences (e.g.,at least 2-fold over background). Stringent conditions aresequence-dependent and will be different in different circumstances. Bycontrolling the stringency of the hybridization and/or washingconditions, target sequences that are 100% complementary to the probecan be identified (homologous probing). Alternatively, stringencyconditions can be adjusted to allow some mismatching in sequences sothat lower degrees of identity are detected (heterologous probing).Generally, a probe is less than about 1000 nucleotides in length or lessthan 500 nucleotides in length.

As used herein, a substantially identical or complementary sequence is apolynucleotide that will specifically hybridize to the complement of thenucleic acid molecule to which it is being compared under highstringency conditions. Appropriate stringency conditions which promoteDNA hybridization, for example, 6× sodium chloride/sodium citrate (SSC)at about 45° C., followed by a wash of 2×SSC at 50° C., are known tothose skilled in the art or can be found in Current Protocols inMolecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.Typically, stringent conditions for hybridization and detection will bethose in which the salt concentration is less than about 1.5 M Na ion,typically about 0.01 to 1.0 M Na ion concentration (or other salts) atpH 7.0 to 8.3 and the temperature is at least about 30° C. for shortprobes (e.g., 10 to 50 nucleotides) and at least about 60° C. for longprobes (e.g., greater than 50 nucleotides). Stringent conditions mayalso be achieved with the addition of destabilizing agents such asformamide. Exemplary low stringency conditions include hybridizationwith a buffer solution of 30 to 35% formamide, 1 M NaCl, 1% SDS (sodiumdodecyl sulphate) at 37° C., and a wash in 1× to 2×SSC (20×SSC=3.0 MNaCl/0.3 M trisodium citrate) at 50 to 55° C. Exemplary moderatestringency conditions include hybridization in 40 to 45% formamide, 1.0M NaCl, 1% SDS at 37° C., and a wash in 0.5× to 1×SSC at 55 to 60° C.Exemplary high stringency conditions include hybridization in 50%formamide, 1 M NaCl, 1% SDS at 37° C., and a wash in 0.1×SSC at 60 to65° C. Optionally, wash buffers may comprise about 0.1% to about 1% SDS.Duration of hybridization is generally less than about 24 hours, usuallyabout 4 to about 12 hours. The duration of the wash time will be atleast a length of time sufficient to reach equilibrium.

In hybridization reactions, specificity is typically the function ofpost-hybridization washes, the critical factors being the ionic strengthand temperature of the final wash solution. For DNA-DNA hybrids, theT_(m) can be approximated from the equation of Meinkoth and Wahl (1984)Anal. Biochem. 138:267-284: T_(m)=81.5° C.+16.6 (log M)+0.41 (% GC)−0.61(% form)−500/L; where M is the molarity of monovalent cations, % GC isthe percentage of guanosine and cytosine nucleotides in the DNA, % formis the percentage of formamide in the hybridization solution, and L isthe length of the hybrid in base pairs. The T_(m) is the temperature(under defined ionic strength and pH) at which 50% of a complementarytarget sequence hybridizes to a perfectly matched probe. T_(m) isreduced by about 1° C. for each 1% of mismatching; thus, T_(m),hybridization, and/or wash conditions can be adjusted to hybridize tosequences of the desired identity. For example, if sequences with ≧90%identity are sought, the T_(m) can be decreased 10° C. Generally,stringent conditions are selected to be about 5° C. lower than thethermal melting point (T_(m)) for the specific sequence and itscomplement at a defined ionic strength and pH. However, severelystringent conditions can utilize a hybridization and/or wash at 1, 2, 3,or 4° C. lower than the thermal melting point (T_(m)); moderatelystringent conditions can utilize a hybridization and/or wash at 6, 7, 8,9, or 10° C. lower than the thermal melting point (T_(m)); lowstringency conditions can utilize a hybridization and/or wash at 11, 12,13, 14, 15, or 20° C. lower than the thermal melting point (T_(m)).Using the equation, hybridization and wash compositions, and desiredT_(m), those of ordinary skill will understand that variations in thestringency of hybridization and/or wash solutions are inherentlydescribed. If the desired degree of mismatching results in a T_(m) ofless than 45° C. (aqueous solution) or 32° C. (formamide solution), itis optimal to increase the SSC concentration so that a highertemperature can be used. An extensive guide to the hybridization ofnucleic acids is found in Tijssen (1993) Laboratory Techniques inBiochemistry and Molecular Biology—Hybridization with Nucleic AcidProbes, Part I, Chapter 2 (Elsevier, N.Y.); and Ausubel et al., eds.(1995) Current Protocols in Molecular Biology, Chapter 2 (GreenePublishing and Wiley-Interscience, New York). See Sambrook et al. (1989)Molecular Cloning: A Laboratory Manual (2d ed., Cold Spring HarborLaboratory Press, Plainview, N.Y.) and Haymes et al. (1985) In: NucleicAcid Hybridization, a Practical Approach, IRL Press, Washington, D.C.

A polynucleotide is said to be the “complement” of anotherpolynucleotide if they exhibit complementarity. As used herein,molecules are said to exhibit “complete complementarity” when everynucleotide of one of the polynucleotide molecules is complementary to anucleotide of the other. Two molecules are said to be “minimallycomplementary” if they can hybridize to one another with sufficientstability to permit them to remain annealed to one another under atleast conventional “low-stringency” conditions. Similarly, the moleculesare said to be “complementary” if they can hybridize to one another withsufficient stability to permit them to remain annealed to one anotherunder conventional “high-stringency” conditions.

Various methods of detection include, but are not limited to, GeneticBit Analysis (Nikiforov et al. (1994) Nucleic Acid Res. 22: 4167-4175)where a DNA oligonucleotide is designed which overlaps both the adjacentflanking DNA sequence and the inserted DNA sequence. The oligonucleotideis immobilized in wells of a microwell plate. Following PCR of theregion of interest (using one primer in the inserted sequence and one inthe adjacent flanking sequence) a single-stranded PCR product can behybridized to the immobilized oligonucleotide and serve as a templatefor a single base extension reaction using a DNA polymerase and labeledddNTPs specific for the expected next base. Readout may be fluorescentor ELISA-based. A signal indicates presence of the insert/flankingsequence due to successful amplification, hybridization, and single baseextension.

Another detection method is the Pyrosequencing technique as described byWinge ((2000) Innov. Pharma. Tech. 00: 18-24). In this method, anoligonucleotide is designed that overlaps the adjacent DNA and insertDNA junction. The oligonucleotide is hybridized to a single-stranded PCRproduct from the region of interest (one primer in the inserted sequenceand one in the flanking sequence) and incubated in the presence of a DNApolymerase, ATP, sulfurylase, luciferase, apyrase, adenosine 5′phosphosulfate and luciferin. dNTPs are added individually and theincorporation results in a light signal which is measured. A lightsignal indicates the presence of the transgene insert/flanking sequencedue to successful amplification, hybridization, and single or multi-baseextension.

Fluorescence Polarization as described by Chen et al. ((1999) GenomeRes. 9: 492-498, 1999) is also a method that can be used to detect anamplicon of the invention. Using this method, an oligonucleotide isdesigned which overlaps the flanking and inserted DNA junction. Theoligonucleotide is hybridized to a single-stranded PCR product from theregion of interest (one primer in the inserted DNA and one in theflanking DNA sequence) and incubated in the presence of a DNA polymeraseand a fluorescent-labeled ddNTP. Single base extension results inincorporation of the ddNTP. Incorporation can be measured as a change inpolarization using a fluorometer. A change in polarization indicates thepresence of the transgene insert/flanking sequence due to successfulamplification, hybridization, and single base extension.

Taqman® (PE Applied Biosystems, Foster City, Calif.) is described as amethod of detecting and quantifying the presence of a DNA sequence andis fully understood in the instructions provided by the manufacturer.Briefly, a FRET oligonucleotide probe is designed which overlaps theflanking and insert DNA junction. The FRET probe and PCR primers (oneprimer in the insert DNA sequence and one in the flanking genomicsequence) are cycled in the presence of a thermostable polymerase anddNTPs. Hybridization of the FRET probe results in cleavage and releaseof the fluorescent moiety away from the quenching moiety on the FRETprobe. A fluorescent signal indicates the presence of theflanking/transgene insert sequence due to successful amplification andhybridization.

Molecular Beacons have been described for use in sequence detection asdescribed in Tyangi et al. ((1996) Nature Biotech. 14: 303-308).Briefly, a FRET oligonucleotide probe is designed that overlaps theflanking and insert DNA junction. The unique structure of the FRET proberesults in it containing secondary structure that keeps the fluorescentand quenching moieties in close proximity. The FRET probe and PCRprimers (one primer in the insert DNA sequence and one in the flankingsequence) are cycled in the presence of a thermostable polymerase anddNTPs. Following successful PCR amplification, hybridization of the FRETprobe to the target sequence results in the removal of the probesecondary structure and spatial separation of the fluorescent andquenching moieties. A fluorescent signal results. A fluorescent signalindicates the presence of the flanking/transgene insert sequence due tosuccessful amplification and hybridization.

A hybridization reaction using a probe specific to a sequence foundwithin the amplicon is yet another method used to detect the ampliconproduced by a PCR reaction.

As used herein, “kit” refers to a set of reagents for the purpose ofperforming the various method of detecting or identifying providedherein, more particularly, the identification and/or the detection of asoybean plant having an enhance resistance to cyst nematode.

Once the soybean plant with a duplication of a region within the rhg1locus conferring resistance to cyst nematode has been identified, theplant or any one of its progeny having this region can be selected andcrossed with a second soybean plant. In specific embodiments, theduplication of a region within the rhg1 locus conferring resistance tocyst nematode can be introgressed into a second soybean plant to producean introgressed soybean germplasm.

V. Transgenic Plants Having an Enhanced Resistance to Cyst Nematode

A transgenic approach can be used to generate additional cyst nematoderesistant materials. Specifically, transgenic integration of one or moreof the genes contained within the duplicated region of the rhg1 locus(for example, one or more of the genes contained between about genomicposition GM18:1663448 and about position GM18:1632228) can be introducedinto a plant or plant cell and expressed and thereby confer enhancedresistance to cyst nematodes. Thus, plants, plant cells and plant partshaving an increased level of expression of one or more genes foundbetween about genomic position GM18:1663448 and about positionGM18:1632228 are provided.

In specific embodiments, a plant, plant cell, seed, grain or plant part(particularly a soybean plant, plant cell, seed or grain) is providedcomprising at least one heterologous polynucleotide stably incorporatedin the genome comprising at least gene found between about genomicposition GM18:1663448 and about position GM18:1632228. In specificembodiments, the heterologous polynucleotide comprises at least one ofthe sequences of Glyma18g2580 (SEQ ID NO: 3), Glyma18g2590 (SEQ IDNO:2), Glyma18g2600 (SEQ ID NO:4), Glyma18g2610 (SEQ ID NO:5), orGlyma18g2570 (SEQ ID NO:1), or an active variant or fragment thereof.The active variant or fragment of the gene will continue to conferenhanced resistance to cyst nematodes.

In addition, while any combination of SEQ ID NO: 1, 2, 3, 4, or 5 oractive variants or fragments thereof can be introduced into the plant orplant part, the plant or plant part can further comprise multiple copiesof the same heterologous polynucleotide. For example, the plant cancomprise at least 2, 3, 4, 5, or more copies of any one of, or anycombination of, Glyma18g2580 (SEQ ID NO: 3), Glyma18g2590 (SEQ ID NO:2),Glyma18g2600 (SEQ ID NO:4), Glyma18g2610 (SEQ ID NO:5), or Glyma18g2570(SEQ ID NO:1), or an active variant or fragment thereof.

In specific embodiments, the heterologous polynucleotide of SEQ ID NO:1, 2, 3, 4 or 5 or the active variant or fragment thereof is operablylinked to a constitutive, tissue-preferred, or other promoter forexpression in plants. In specific embodiments the promoter isheterologous to the polynucleotide of SEQ ID NO: 1, 2, 3, 4 or 5.

In specific embodiments the plant or plant cell having the heterologouspolynucleotide is a soybean plant. However, the sequence can beintroduced into any plant of interest, including, but not limited to,monocots and dicots. Examples of plant species of interest include, butare not limited to, corn (Zea mays), Brassica sp. (e.g., B. napus, B.rapa, B. juncea), particularly those Brassica species useful as sourcesof seed oil, alfalfa (Medicago sativa), rice (Oryza sativa), rye (Secalecereale), sorghum (Sorghum bicolor, Sorghum vulgare), millet (e.g.,pearl millet (Pennisetum glaucum), proso millet (Panicum miliaceum),foxtail millet (Setaria italica), finger millet (Eleusine coracana)),sunflower (Helianthus annuus), safflower (Carthamus tinctorius), wheat(Triticum aestivum), soybean (Glycine max), tobacco (Nicotiana tabacum),potato (Solanum tuberosum), peanuts (Arachis hypogaea), cotton(Gossypium barbadense, Gossypium hirsutum), sweet potato (Ipomoeabatatus), cassava (Manihot esculenta), coffee (Coffea spp.), coconut(Cocos nucifera), pineapple (Ananas comosus), citrus trees (Citrusspp.), cocoa (Theobroma cacao), tea (Camellia sinensis), banana (Musaspp.), avocado (Persea americana), fig (Ficus casica), guava (Psidiumguajava), mango (Mangifera indica), olive (Olea europaea), papaya(Carica papaya), cashew (Anacardium occidentale), macadamia (Macadamiaintegrifolia), almond (Prunus amygdalus), sugar beets (Beta vulgaris),sugarcane (Saccharum spp.), oats, barley, vegetables, ornamentals, andconifers.

Vegetables include tomatoes (Lycopersicon esculentum), lettuce (e.g.,Lactuca sativa), green beans (Phaseolus vulgaris), lima beans (Phaseoluslimensis), peas (Lathyrus spp.), and members of the genus Cucumis suchas cucumber (C. sativus), cantaloupe (C. cantalupensis), and musk melon(C. melo). Ornamentals include azalea (Rhododendron spp.), hydrangea(Macrophylla hydrangea), hibiscus (Hibiscus rosasanensis), roses (Rosaspp.), tulips (Tulipa spp.), daffodils (Narcissus spp.), petunias(Petunia hybrida), carnation (Dianthus caryophyllus), poinsettia(Euphorbia pulcherrima), and chrysanthemum.

Conifers that may be employed in practicing the present inventioninclude, for example, pines such as loblolly pine (Pinus taeda), slashpine (Pinus elliotii), ponderosa pine (Pinus ponderosa), lodgepole pine(Pinus contorta), and Monterey pine (Pinus radiata); Douglas-fir(Pseudotsuga menziesii); Western hemlock (Tsuga canadensis); Sitkaspruce (Picea glauca); redwood (Sequoia sempervirens); true firs such assilver fir (Abies amabilis) and balsam fir (Abies balsamea); and cedarssuch as Western red cedar (Thuja plicata) and Alaska yellow-cedar(Chamaecyparis nootkatensis), and Poplar and Eucalyptus. In specificembodiments, plants of the present invention are crop plants (forexample, corn, alfalfa, sunflower, Brassica, soybean, cotton, safflower,peanut, sorghum, wheat, millet, tobacco, etc.). In other embodiments,corn and soybean plants are optimal, and in yet other embodiments cornplants are optimal.

Other plants of interest include grain plants that provide seeds ofinterest, oil-seed plants, and leguminous plants. Seeds of interestinclude grain seeds, such as corn, wheat, barley, rice, sorghum, rye,etc. Oil-seed plants include cotton, soybean, safflower, sunflower,Brassica, maize, alfalfa, palm, coconut, etc. Leguminous plants includebeans and peas. Beans include guar, locust bean, fenugreek, soybean,garden beans, cowpea, mungbean, lima bean, fava bean, lentils, chickpea,etc.

A. Variants and Fragments of Glyma18g2580, Glyma18g2590, Glyma18g2600,Glyma18g2610, and Glyma18g2570

“Variants” is intended to mean substantially similar sequences. Forpolynucleotides, a variant comprises a polynucleotide having a deletion(i.e., truncations) at the 5′ and/or 3′ end and/or a deletion and/oraddition of one or more nucleotides at one or more internal sites withinthe native polynucleotide and/or a substitution of one or morenucleotides at one or more sites in the native polynucleotide. As usedherein, a “native” polynucleotide or polypeptide comprises a naturallyoccurring nucleotide sequence or amino acid sequence, respectively. Forpolynucleotides, conservative variants include those sequences that,because of the degeneracy of the genetic code, encode the amino acidsequence of one of the polypeptides found between about genomic positiongenomic position GM18:1663448 and about position GM18:1632228,particularly, SEQ ID NO: 1, 2, 3, 4, or 5. Variant polynucleotides alsoinclude synthetically derived polynucleotides, such as those generated,for example, by using site-directed mutagenesis or gene synthesis butwhich still retain the ability to enhance resistance to cyst nematodes.

An active variant of any one of SEQ ID NO: 1, 2, 3, 4, or 5 can comprisea polynucleotide having at least 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%,94%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 1, 2, 3,4, or 5, as determined by sequence alignment programs and parametersdescribed elsewhere herein, and when expressed, the sequence continue toconfer enhanced resistance to cyst nematodes. Non-limiting examples ofvariants of SEQ ID NO: 1, 2, 3, and 5 are set forth in Table 2.

Obviously, the mutations that will be made in the DNA encoding thevariant must not place the sequence out of reading frame and optimallywill not create complementary regions that could produce secondary mRNAstructure. See, EP Patent Application Publication No. 75,444. Variantpolynucleotides and proteins also encompass sequences and proteinsderived from a mutagenic and recombinogenic procedure such as DNAshuffling. Strategies for such DNA shuffling are known in the art. See,for example, Stemmer (1994) Proc. Natl. Acad Sci. USA 91:10747-10751;Stemmer (1994) Nature 370:389-391; Crameri et al. (1997) Nature Biotech.15:436-438; Moore et al. (1997) J. Mol. Biol. 272:336-347; Zhang et al.(1997) Proc. Natl. Acad. Sci. USA 94:4504-4509; Crameri et al. (1998)Nature 391:288-291; and U.S. Pat. Nos. 5,605,793 and 5,837,458.

By “fragment” is intended a portion of the polynucleotide or a portionof the amino acid sequence and hence protein encoded thereby. Fragmentsof a polynucleotide may encode protein fragments that retain the abilityto enhance resistance to nematodes. Fragments of a nucleotide sequencemay range from at least about 20 nucleotides, about 50 nucleotides,about 100 nucleotides, and up to the full-length polynucleotide encodingthe polypeptides that confer enhanced resistance to cyst nematodes. Afragment of a SEQ ID NO: 1, 2, 3, 4 or 5 polynucleotide that encodes abiologically active portion and thereby enhances resistance to cystnematodes will comprise at least 50, 75, 100, 150, 175, 200, 225, 250,275, 300, 325, 350, 375, 400, 410, 415, 420, 425, 430, 435, or 440contiguous nucleotides, or up to the total number of nucleotides presentin a full-length sequences.

B. Polynucleotide Constructs

The polynucleotides disclosed herein that confer enhanced resistance tocyst nematodes (i.e., SEQ ID NO: 1, 2, 3, 4, or 5 or active variants andfragments thereof) can be provided in expression cassettes forexpression in the plant of interest. The cassette can include 5′ and 3′regulatory sequences operably linked to the polynucleotide or activevariant or fragment thereof conferring enhanced resistance to cyctnematodes. “Operably linked” is intended to mean a functional linkagebetween two or more elements. For example, an operable linkage between apolynucleotide of interest and a regulatory sequence (i.e., a promoter)is a functional link that allows for expression of the polynucleotide ofinterest. Operably linked elements may be contiguous or non-contiguous.When used to refer to the joining of two protein coding regions, byoperably linked is intended that the coding regions are in the samereading frame. The cassette may additionally contain at least oneadditional gene to be cotransformed into the organism. Alternatively,the additional gene(s) can be provided on multiple expression cassettes.Such an expression cassette is provided with a plurality of restrictionsites and/or recombination sites for insertion of the polynucleotide oractive variant or fragment thereof to be under the transcriptionalregulation of the regulatory regions. The expression cassette mayadditionally contain selectable marker genes.

The expression cassette can include in the 5′-3′ direction oftranscription, a transcriptional and translational initiation region(i.e., a promoter), a polynucleotide or active variant or fragmentthereof conferring enhanced resistance to cyst nematode (i.e., SEQ IDNO: 1, 2, 3, 4 or 5), and a transcriptional and translationaltermination region (i.e., termination region) functional in plants. Theregulatory regions (i.e., promoters, transcriptional regulatory regions,and translational termination regions) and/or the polynucleotide oractive variant or fragment thereof may be native/analogous to the hostcell or to each other. Alternatively, the regulatory regions and/or thesequences conferring enhanced resistance to cyst nematode of or activevariant or fragment thereof may be heterologous to the host cell or toeach other.

As used herein, “heterologous” in reference to a sequence is a sequencethat originates from a foreign species, or, if from the same species, issubstantially modified from its native form in composition and/orgenomic locus by deliberate human intervention. For example, a promoteroperably linked to a heterologous polynucleotide is from a speciesdifferent from the species from which the polynucleotide was derived,or, if from the same/analogous species, one or both are substantiallymodified from their original form and/or genomic locus, or the promoteris not the native promoter for the operably linked polynucleotide.

As used herein, polynucleotide or polypeptide is “recombinant” when itis artificial or engineered, or derived from an artificial or engineeredprotein or nucleic acid. For example, a polynucleotide that is insertedinto a vector or any other heterologous location, e.g., in a genome of arecombinant organism, such that it is not associated with nucleotidesequences that normally flank the polynucleotide as it is found innature is a recombinant polynucleotide. A polypeptide expressed in vitroor in vivo from a recombinant polynucleotide is an example of arecombinant polypeptide. Likewise, a polynucleotide sequence that doesnot appear in nature, for example, a variant of a naturally occurringgene is recombinant.

The termination region may be native with the transcriptional initiationregion or active variant or fragment thereof, may be native with theplant host, or may be derived from another source (i.e., foreign orheterologous) to the promoter, the polynucleotide or active fragment orvariant thereof encoding the polypeptide enhancing resistance to cystnematode, the plant host, or any combination thereof. Convenienttermination regions are available from the Ti-plasmid of A. tumefaciens,such as the octopine synthase and nopaline synthase termination regions.See also Guerineau et al. (1991) Mol. Gen. Genet. 262:141-144; Proudfoot(1991) Cell 64:671-674; Sanfacon et al. (1991) Genes Dev. 5:141-149;Mogen et al. (1990) Plant Cell 2:1261-1272; Munroe et al. (1990) Gene91:151-158; Ballas et al. (1989) Nucleic Acids Res. 17:7891-7903; andJoshi et al. (1987) Nucleic Acids Res. 15:9627-9639.

Where appropriate, the polynucleotides may be optimized for increasedexpression in the transformed plant. That is, the polynucleotides can besynthesized using plant-preferred codons for improved expression. See,for example, Campbell and Gowri (1990) Plant Physiol. 92:1-11 for adiscussion of host-preferred codon usage. Methods are available in theart for synthesizing plant-preferred genes. See, for example, U.S. Pat.Nos. 5,380,831, and 5,436,391, and Murray et al. (1989) Nucleic AcidsRes. 17:477-498, herein incorporated by reference.

Additional sequence modifications are known to enhance gene expressionin a cellular host. These include elimination of sequences encodingspurious polyadenylation signals, exon-intron splice site signals,transposon-like repeats, and other such well-characterized sequencesthat may be deleterious to gene expression. The G-C content of thesequence may be adjusted to levels average for a given cellular host, ascalculated by reference to known genes expressed in the host cell. Whenpossible, the sequence is modified to avoid predicted hairpin secondarymRNA structures.

The expression cassettes may additionally contain 5′ leader sequences.Such leader sequences can act to enhance translation. Translationleaders are known in the art and include: picornavirus leaders, forexample, EMCV leader (Encephalomyocarditis 5′ noncoding region)(Elroy-Stein et al. (1989) Proc. Natl. Acad. Sci. USA 86:6126-6130);potyvirus leaders, for example, TEV leader (Tobacco Etch Virus) (Gallieet al. (1995) Gene 165(2):233-238), MDMV leader (Maize Dwarf MosaicVirus) (Virology 154:9-20), and human immunoglobulin heavy-chain bindingprotein (BiP) (Macejak et al. (1991) Nature 353:90-94); untranslatedleader from the coat protein mRNA of alfalfa mosaic virus (AMV RNA 4)(Jobling et al. (1987) Nature 325:622-625); tobacco mosaic virus leader(TMV) (Gallie et al. (1989) in Molecular Biology of RNA, ed. Cech (Liss,New York), pp. 237-256); and maize chlorotic mottle virus leader (MCMV)(Lommel et al. (1991) Virology 81:382-385. See also, Della-Cioppa et al.(1987) Plant Physiol. 84:965-968.

In preparing the expression cassette, the various DNA fragments may bemanipulated, so as to provide for the DNA sequences in the properorientation and, as appropriate, in the proper reading frame. Towardthis end, adapters or linkers may be employed to join the DNA fragmentsor other manipulations may be involved to provide for convenientrestriction sites, removal of superfluous DNA, removal of restrictionsites, or the like. For this purpose, in vitro mutagenesis, primerrepair, restriction, annealing, resubstitutions, e.g., transitions andtransversions, may be involved.

A number of promoters can be used to express the various sequence setforth in SEQ ID NO:1, 2, 3, 4, or 5 or the active variant or fragmentsthere, including the native promoter of the polynucleotide sequence ofinterest. The promoters can be selected based on the desired outcome.Such promoters include, for example, constitutive, inducible,tissue-preferred, or other promoters for expression in plants or in anyorganism of interest. In specific embodiments, the promoters areheterologous to the sequences being expressed.

Constitutive promoters include, for example, the core promoter of theRsyn7 promoter and other constitutive promoters disclosed in WO 99/43838and U.S. Pat. No. 6,072,050; the core CaMV 35S promoter (Odell et al.(1985) Nature 313:810-812); rice actin (McElroy et a (1990) Plant Cell2:163-171); ubiquitin (Christensen et al. (1989) Plant Mol. Biol.12:619-632 and Christensen et al. (1992) Plant Mol. Biol. 18:675-689);pEMU (Last et al. (1991) Theor. Appl. Genet. 81:581-588); MAS (Velten etal. (1984) EMBO J. 3:2723-2730); ALS promoter (U.S. Pat. No. 5,659,026),and the like. Other constitutive promoters include, for example, U.S.Pat. Nos. 5,608,149; 5,608,144; 5,604,121; 5,569,597; 5,466,785;5,399,680; 5,268,463; 5,608,142; and 6,177,611.

Tissue-preferred promoters can be utilized to target expression within aparticular plant tissue. Tissue-preferred promoters include thosedescribed in Yamamoto et al. (1997) Plant J. 12(2):255-265; Kawamata etal. (1997) Plant Cell Physiol. 38(7):792-803; Hansen et al. (1997) Mol.Gen Genet. 254(3):337-343; Russell et al. (1997) Transgenic Res.6(2):157-168; Rinehart et al. (1996) Plant Physiol. 112(3):1331-1341;Van Camp et a (1996) Plant Physiol. 112(2):525-535; Canevascini et al.(1996) Plant Physiol. 112(2):513-524; Yamamoto et al. (1994) Plant CellPhysiol. 35(5):773-778; Lam (1994) Results Probl. Cell Differ.20:181-196; Orozco et al. (1993) Plant Mol Biol. 23(6):1129-1138;Matsuoka et al. (1993) Proc Natl. Acad. Sci. USA 90(20):9586-9590; andGuevara-Garcia et al. (1993) Plant J. 4(3):495-505. Such promoters canbe modified, if necessary, for weak expression. An exemplary promoter isthe anther specific promoter 5126 (U.S. Pat. Nos. 5,689,049 and5,689,051). Examples of seed-preferred promoters include, but are notlimited to, 27 kD gamma zein promoter and waxy promoter, Boronat et al.Plant Sci. 47, 95-102 (1986); Reina et al. Nucleic Acids Res. 18 (21),6426 (1990); and Kloesgen et al., Mol. Gen. Genet. 203, 237-244 (1986).Promoters that express in the embryo, pericarp, and endosperm aredisclosed in U.S. Patent Application Ser. Nos. 60/097,233 filed Aug. 20,1998 and 60/098,230 filed Aug. 28, 1998. The disclosures each of theseare incorporated herein by reference in their entirety.

Leaf-preferred promoters are known in the art. See, for example,Yamamoto et al. (1997) Plant J. 12(2):255-265; Kwon et al. (1994) PlantPhysiol. 105:357-67; Yamamoto et al. (1994) Plant Cell Physiol.35(5):773-778; Gotor et al. (1993) Plant J. 3:509-18; Orozco et al.(1993) Plant Mol. Biol. 23(6):1129-1138; and Matsuoka et al. (1993)Proc. Natl. Acad. Sci. USA 90(20):9586-9590.

Synthetic promoters can be used to express the various sequences thatconfer tolerance to cyst nematodes or biologically active variants andfragments thereof.

Chemical-regulated promoters can be used to modulate the expression of agene in a plant through the application of an exogenous chemicalregulator. Depending upon the objective, the promoter may be achemical-inducible promoter, where application of the chemical inducesgene expression, or a chemical-repressible promoter, where applicationof the chemical represses gene expression. Chemical-inducible promotersare known in the art and include, but are not limited to, the maizeIn2-2 promoter, which is activated by benzenesulfonamide herbicidesafeners; the maize GST promoter, which is activated by hydrophobicelectrophilic compounds that are used as pre-emergent herbicides; andthe tobacco PR-1a promoter, which is activated by salicylic acid. Otherchemical-regulated promoters of interest include steroid-responsivepromoters. See, for example, the glucocorticoid-inducible promoter inSchena et al. (1991) Proc. Natl. Acad. Sci. USA 88:10421-10425 andMcNellis et al. (1998) Plant J. 14(2):247-257 and thetetracycline-inducible and tetracycline-repressible promoters forexample, Gatz et al. (1991) Mol. Gen. Genet. 227:229-237, and U.S. Pat.Nos. 5,814,618 and 5,789,156, herein incorporated by reference.

The expression cassette can also comprise a selectable marker gene forthe selection of transformed cells. Selectable marker genes are utilizedfor the selection of transformed cells or tissues. Marker genes includegenes encoding antibiotic resistance, such as those encoding neomycinphosphotransferase II (NEO) and hygromycin phosphotransferase (HPT), aswell as genes conferring resistance to herbicidal compounds, such asglyphosate, glufosinate ammonium, bromoxynil, sulfonylureas, dicamba,and 2,4-dichlorophenoxyacetate (2,4-D). Additional selectable markersinclude phenotypic markers such as β-galactosidase and fluorescentproteins such as green fluorescent protein (GFP) (Su et al. (2004)Biotechnol Bioeng 85:610-9 and Fetter et al. (2004) Plant Cell16:215-28), cyan florescent protein (CYP) (Bolte et al. (2004) J. CellScience 117:943-54 and Kato et al. (2002) Plant Physiol 129:913-42), andyellow florescent protein (PhiYFP™ from Evrogen, see, Bolte et al.(2004) J. Cell Science 117:943-54). For additional selectable markers,see generally, Yarranton (1992) Curr. Opin. Biotech. 3:506-511;Christopherson et al. (1992) Proc. Natl. Acad. Sci. USA 89:6314-6318;Yao et al. (1992) Cell 71:63-72; Reznikoff (1992) Mol. Microbiol.6:2419-2422; Barkley et al. (1980) in The Operon, pp. 177-220; Hu et al.(1987) Cell 48:555-566; Brown et al. (1987) Cell 49:603-612; Figge etal. (1988) Cell 52:713-722; Deuschle et al. (1989) Proc. Natl. Acad.Aci. USA 86:5400-5404; Fuerst et al. (1989) Proc. Natl. Acad. Sci. USA86:2549-2553; Deuschle et al. (1990) Science 248:480-483; Gossen (1993)Ph.D. Thesis, University of Heidelberg; Reines et al. (1993) Proc. Natl.Acad. Sci. USA 90:1917-1921; Labow et al. (1990) Mol. Cell. Biol.10:3343-3356; Zambretti et al. (1992) Proc. Natl. Acad. Sci. USA89:3952-3956; Baim et al. (1991) Proc. Natl. Acad. Sci. USA88:5072-5076; Wyborski et al. (1991) Nucleic Acids Res. 19:4647-4653;Hillenand-Wissman (1989) Topics Mol. Struc. Biol. 10:143-162; Degenkolbet al. (1991) Antimicrob. Agents Chemother. 35:1591-1595; Kleinschnidtet al. (1988) Biochemistry 27:1094-1104; Bonin (1993) Ph.D. Thesis,University of Heidelberg; Gossen et al. (1992) Proc. Natl. Acad. Sci.USA 89:5547-5551; Oliva et al. (1992) Antimicrob. Agents Chemother.36:913-919; Hlavka et al. (1985) Handbook of Experimental Pharmacology,Vol. 78 (Springer-Verlag, Berlin); Gill et al. (1988) Nature334:721-724. Such disclosures are herein incorporated by reference. Theabove list of selectable marker genes is not meant to be limiting. Anyselectable marker gene can be used in the present invention, includingfor example, DsRed.

C. Stacking Other Traits of Interest

In some embodiments, the polynucleotides conferring enhanced toleranceto cyst nematodes or active variants and fragments thereof areengineered into a molecular stack. Thus, the various plants, plant cellsand seeds disclosed herein can further comprise one or more traits ofinterest, and in more specific embodiments, the host cell, plant, plantpart or plant cell is stacked with any combination of polynucleotidesequences of interest in order to create plants with a desiredcombination of traits. As used herein, the term “stacked” includeshaving the multiple traits present in the same plant or organism ofinterest. In one non-limiting example, “stacked traits” comprise amolecular stack where the sequences are physically adjacent to eachother. A trait, as used herein, refers to the phenotype derived from aparticular sequence or groups of sequences.

The plant or plant cell or plant part having the sequence conferringenhanced tolerance to cyst nematodes or active variants or fragmentsthereof can also be combined with at least one other trait to produceplants that further comprise a variety of desired trait combinationsincluding, but not limited to, traits desirable for animal feed such ashigh oil content (e.g., U.S. Pat. No. 6,232,529); balanced amino acidcontent (e.g., hordothionins (U.S. Pat. Nos. 5,990,389; 5,885,801;5,885,802; and 5,703,409; U.S. Pat. No. 5,850,016); barley high lysine(Williamson et al. (1987) Eur. J. Biochem. 165: 99-106; and WO 98/20122)and high methionine proteins (Pedersen et al. (1986) J. Biol. Chem. 261:6279; Kirihara et al. (1988) Gene 71: 359; and Musumura et al. (1989)Plant Mol. Biol. 12:123)); increased digestibility (e.g., modifiedstorage proteins (U.S. application Ser. No. 10/053,410, filed Nov. 7,2001); and thioredoxins (U.S. application Ser. No. 10/005,429, filedDec. 3, 2001)); the disclosures of which are herein incorporated byreference. Desired trait combinations also include LLNC (low linolenicacid content; see, e.g., Dyer et al. (2002) Appl. Microbiol. Biotechnol.59: 224-230) and OLCH (high oleic acid content; see, e.g.,Fernandez-Moya et al. (2005) J. Agric. Food Chem. 53: 5326-5330).

The plant or plant cell or plant part having the sequence or an activevariant or fragment thereof which confers enhanced resistance to cystnematodes can also be combined with other desirable traits such as, forexample, fumonisim detoxification genes (U.S. Pat. No. 5,792,931),avirulence and disease resistance genes (Jones et al. (1994) Science266: 789; Martin et al. (1993) Science 262: 1432; Mindrinos et al.(1994) Cell 78: 1089), and traits desirable for processing or processproducts such as modified oils (e.g., fatty acid desaturase genes (U.S.Pat. No. 5,952,544; WO 94/11516)); modified starches (e.g., ADPGpyrophosphorylases (AGPase), starch synthases (SS), starch branchingenzymes (SBE), and starch debranching enzymes (SDBE)); and polymers orbioplastics (e.g., U.S. Pat. No. 5,602,321; beta-ketothiolase,polyhydroxybutyrate synthase, and acetoacetyl-CoA reductase (Schubert etal. (1988) J. Bacteriol. 170:5837-5847) facilitate expression ofpolyhydroxyalkanoates (PHAs)); the disclosures of which are hereinincorporated by reference. One could also combine herbicide-tolerantpolynucleotides with polynucleotides providing agronomic traits such asmale sterility (e.g., see U.S. Pat. No. 5,583,210), stalk strength,flowering time, or transformation technology traits such as cell cycleregulation or gene targeting (e.g., WO 99/61619, WO 00/17364, and WO99/25821); the disclosures of which are herein incorporated byreference.

In other embodiments, the plant or plant cell or plant part having thesequence that confers enhanced resistance to cyst nematode or an activevariant or fragment thereof may be stacked with any otherpolynucleotides encoding polypeptides having pesticidal and/orinsecticidal activity, such as Bacillus thuringiensis toxic proteins(described in U.S. Pat. Nos. 5,366,892; 5,747,450; 5,737,514; 5,723,756;5,593,881; Geiser et al. (1986) Gene 48: 109; Lee et al. (2003) Appl.Environ. Microbiol. 69: 4648-4657 (Vip3A); Galitzky et al. (2001) ActaCrystallogr. D. Biol. Crystallogr. 57: 1101-1109 (Cry3Bb1); and Hermanet al. (2004) J. Agric. Food Chem. 52: 2726-2734 (Cry1F)), lectins (VanDamme et al. (1994) Plant Mol. Biol. 24: 825, pentin (described in U.S.Pat. No. 5,981,722), and the like. The combinations generated can alsoinclude multiple copies of any one of the polynucleotides of interest.

In another embodiment, the plant or plant cell or plant part having thesequence that confers enhanced resistance to cyst nematode or an activevariant or fragment thereof can also be combined with the Rcg1 sequenceor biologically active variant or fragment thereof. The Rcg1 sequence isan anthracnose stalk rot resistance gene in corn. See, for example, U.S.patent application Ser. Nos. 11/397,153, 11/397,275, and 11/397,247,each of which is herein incorporated by reference.

These stacked combinations can be created by any method including, butnot limited to, breeding plants by any conventional methodology, orgenetic transformation. If the sequences are stacked by geneticallytransforming the plants, the polynucleotide sequences of interest can becombined at any time and in any order. The traits can be introducedsimultaneously in a co-transformation protocol with the polynucleotidesof interest provided by any combination of transformation cassettes. Forexample, if two sequences will be introduced, the two sequences can becontained in separate transformation cassettes (trans) or contained onthe same transformation cassette (cis). Expression of the sequences canbe driven by the same promoter or by different promoters. In certaincases, it may be desirable to introduce a transformation cassette thatwill suppress the expression of the polynucleotide of interest. This maybe combined with any combination of other suppression cassettes oroverexpression cassettes to generate the desired combination of traitsin the plant. It is further recognized that polynucleotide sequences canbe stacked at a desired genomic location using a site-specificrecombination system. See, for example, WO99/25821, WO99/25854,WO99/25840, WO99/25855, and WO99/25853, all of which are hereinincorporated by reference.

Any plant having the sequence that confers enhanced resistance to cystnematode disclosed herein or an active variant or fragment thereof canbe used to make a food or a feed product. Such methods compriseobtaining a plant, explant, seed, plant cell, or cell comprising thesequence that confers enhanced resistance to cyst nematode (i.e., SEQ IDNO: 1, 2, 3, 4, or 5) or active variant or fragment thereof andprocessing the plant, explant, seed, plant cell, or cell to produce afood or feed product.

D. Methods of Introducing

Various methods can be used to introduce a sequence of interest into aplant or plant part. “Introducing” is intended to mean presenting to thehost cell, plant, plant cell or plant part the polynucleotide orpolypeptide in such a manner that the sequence gains access to theinterior of a cell of the plant or organism. The methods of theinvention do not depend on a particular method for introducing asequence into an organism or a plant or plant part, only that thepolynucleotide or polypeptides gains access to the interior of at leastone cell of the organism or the plant. Methods for introducingpolynucleotide or polypeptides into various organisms, including plants,are known in the art including, but not limited to, stabletransformation methods, transient transformation methods, andvirus-mediated methods.

“Stable transformation” is intended to mean that the nucleotideconstruct introduced into a plant integrates into the genome of theplant or organism of interest and is capable of being inherited by theprogeny thereof. “Transient transformation” is intended to mean that apolynucleotide is introduced into the plant or organism of interest anddoes not integrate into the genome of the plant or organism or apolypeptide is introduced into a plant or organism.

Transformation protocols as well as protocols for introducingpolypeptides or polynucleotide sequences into plants may vary dependingon the type of plant or plant cell, i.e., monocot or dicot, targeted fortransformation. Suitable methods of introducing polypeptides andpolynucleotides into plant cells include microinjection (Crossway et al.(1986) Biotechniques 4:320-334), electroporation (Riggs et al. (1986)Proc. Natl. Acad. Sci. USA 83:5602-5606, Agrobacterium-mediatedtransformation (U.S. Pat. No. 5,563,055 and U.S. Pat. No. 5,981,840),direct gene transfer (Paszkowski et al. (1984) EMBO J. 3:2717-2722), andballistic particle acceleration (see, for example, U.S. Pat. No.4,945,050; U.S. Pat. No. 5,879,918; U.S. Pat. No. 5,886,244; and, U.S.Pat. No. 5,932,782; Tomes et al. (1995) in Plant Cell, Tissue, and OrganCulture: Fundamental Methods, ed. Gamborg and Phillips (Springer-Verlag,Berlin); McCabe et al. (1988) Biotechnology 6:923-926); and Lec1transformation (WO 00/28058). Also see Weissinger et al. (1988) Ann.Rev. Genet. 22:421-477; Sanford et al. (1987) Particulate Science andTechnology 5:27-37 (onion); Christou et al. (1988) Plant Physiol.87:671-674 (soybean); McCabe et al. (1988) Bio/Technology 6:923-926(soybean); Finer and McMullen (1991) In Vitro Cell Dev. Biol.27P:175-182 (soybean); Singh et al. (1998) Theor. Appl. Genet.96:319-324 (soybean); Datta et al. (1990) Biotechnology 8:736-740(rice); Klein et al. (1988) Proc. Natl. Acad. Sci. USA 85:4305-4309(maize); Klein et al. (1988) Biotechnology 6:559-563 (maize); U.S. Pat.Nos. 5,240,855; 5,322,783; and, 5,324,646; Klein et al. (1988) PlantPhysiol. 91:440-444 (maize); Fromm et al. (1990) Biotechnology 8:833-839(maize); Hooykaas-Van Slogteren et al. (1984) Nature (London)311:763-764; U.S. Pat. No. 5,736,369 (cereals); Bytebier et al. (1987)Proc. Natl. Acad. Sci. USA 84:5345-5349 (Liliaceae); De Wet et al.(1985) in The Experimental Manipulation of Ovule Tissues, ed. Chapman etal. (Longman, N.Y.), pp. 197-209 (pollen); Kaeppler et al. (1990) PlantCell Reports 9:415-418 and Kaeppler et al. (1992) Theor. Appl. Genet.84:560-566 (whisker-mediated transformation); D'Halluin et al. (1992)Plant Cell 4:1495-1505 (electroporation); Li et al. (1993) Plant CellReports 12:250-255 and Christou and Ford (1995) Annals of Botany75:407-413 (rice); Osjoda et al. (1996) Nature Biotechnology 14:745-750(maize via Agrobacterium tumefaciens); all of which are hereinincorporated by reference.

In specific embodiments, the sequences that confer enhanced resistanceto cyst nematodes (i.e., any one or combination of SEQ ID NO: 1, 2, 3,4, or 5) or active variants or fragments thereof can be introduced intoplants by contacting plants with a virus or viral nucleic acids.Generally, such methods involve incorporating a nucleotide construct ofthe invention within a DNA or RNA molecule. It is recognized that thesequence that confers enhanced resistance to cyst nematodes (i.e., anyone or combination of SEQ ID NO: 1, 2, 3, 4, or 5) may be initiallysynthesized as part of a viral polyprotein, which later may be processedby proteolysis in vivo or in vitro to produce the desired recombinantprotein. Further, it is recognized that promoters of the invention alsoencompass promoters utilized for transcription by viral RNA polymerases.Methods for introducing polynucleotides into plants and expressing aprotein encoded therein, involving viral DNA or RNA molecules, are knownin the art. See, for example, U.S. Pat. Nos. 5,889,191, 5,889,190,5,866,785, 5,589,367, 5,316,931, and Porta et al. (1996) MolecularBiotechnology 5:209-221; herein incorporated by reference.

Methods are known in the art for the targeted insertion of apolynucleotide at a specific location in the plant genome. In oneembodiment, the insertion of the polynucleotide at a desired genomiclocation is achieved using a site-specific recombination system. See,for example, WO99/25821, WO99/25854, WO99/25840, WO99/25855, andWO99/25853, all of which are herein incorporated by reference. Briefly,the polynucleotide of the invention can be contained in transfercassette flanked by two non-recombinogenic recombination sites. Thetransfer cassette is introduced into a plant having stably incorporatedinto its genome a target site which is flanked by two non-recombinogenicrecombination sites that correspond to the sites of the transfercassette. An appropriate recombinase is provided and the transfercassette is integrated at the target site. The polynucleotide ofinterest is thereby integrated at a specific chromosomal position in theplant genome. Other methods to target polynucleotides are set forth inWO 2009/114321 (herein incorporated by reference), which describes“custom” meganucleases produced to modify plant genomes, in particularthe genome of maize. See, also, Gao et al. (2010) Plant Journal1:176-187.

The cells that have been transformed may be grown into plants inaccordance with conventional ways. See, for example, McCormick et al.(1986) Plant Cell Reports 5:81-84. These plants may then be grown, andeither pollinated with the same transformed strain or different strains,and the resulting progeny having constitutive expression of the desiredphenotypic characteristic identified. Two or more generations may begrown to ensure that expression of the desired phenotypic characteristicis stably maintained and inherited and then seeds harvested to ensureexpression of the desired phenotypic characteristic has been achieved.In this manner, the present invention provides transformed seed (alsoreferred to as “transgenic seed”) having a polynucleotide of theinvention, for example, an expression cassette of the invention, stablyincorporated into their genome.

Transformed plant cells which are derived by plant transformationtechniques, including those discussed above, can be cultured toregenerate a whole plant which possesses the transformed genotype (i.e.,that confer enhanced resistance to cyst nematodes (i.e., any one orcombination of SEQ ID NO: 1, 2, 3, 4, or 5)), and thus the desiredphenotype, such as enhanced resistance to cyst nematodes. Fortransformation and regeneration of maize see, Gordon-Kamm et al., ThePlant Cell, 2:603-618 (1990). Plant regeneration from culturedprotoplasts is described in Evans et al. (1983) Protoplasts Isolationand Culture, Handbook of Plant Cell Culture, pp 124-176, MacmillanPublishing Company, New York; and Binding (1985) Regeneration of Plants,Plant Protoplasts pp 21-73, CRC Press, Boca Raton. Regeneration can alsobe obtained from plant callus, explants, organs, or parts thereof. Suchregeneration techniques are described generally in Klee et al. (1987)Ann Rev of Plant Phys 38:467. See also, e.g., Payne and Gamborg.

One of skill will recognize that after the expression cassettecontaining the sequence that confer enhanced resistance to cystnematodes (i.e., any one or combination of SEQ ID NO: 1, 2, 3, 4, or 5)is stably incorporated in transgenic plants and confirmed to beoperable, it can be introduced into other plants by sexual crossing. Anyof a number of standard breeding techniques can be used, depending uponthe species to be crossed.

In vegetatively propagated crops, mature transgenic plants can bepropagated by the taking of cuttings or by tissue culture techniques toproduce multiple identical plants. Selection of desirable transgenics ismade and new varieties are obtained and propagated vegetatively forcommercial use. In seed propagated crops, mature transgenic plants canbe self-crossed to produce a homozygous inbred plant. The inbred plantproduces seed containing the newly introduced heterologous nucleic acid.These seeds can be grown to produce plants that would produce theselected phenotype.

Parts obtained from the regenerated plant, such as flowers, seeds,leaves, branches, fruit, and the like are included in the invention,provided that these parts comprise cells comprising the sequence thatconfers enhanced resistance to cyst nematodes (i.e., any one orcombination of SEQ ID NO: 1, 2, 3, 4, or 5). Progeny and variants, andmutants of the regenerated plants are also included within the scope ofthe invention, provided that these parts comprise the introduced nucleicacid sequences.

In one embodiment, a homozygous transgenic plant can be obtained bysexually mating (selfing) a heterozygous transgenic plant that containsa single added heterologous nucleic acid, germinating some of the seedproduced and analyzing the resulting plants produced for altered celldivision relative to a control plant (i.e., native, non-transgenic).Back-crossing to a parental plant and out-crossing with a non-transgenicplant are also contemplated.

E. Methods for Increasing Expression and/or Concentration of at LeastOne Sequence that Confers Enhanced Resistance to Cyst Nematodes in aPlant or Plant Part

A method for increasing the activity and/or concentration of at leastone sequence that confers enhanced resistance to cyst nematodes (i.e.,SEQ ID NO: 1, 2, 3, 4, or 5 or an active variant or fragment thereof) ina plant, plant cell, plant part, explant, and/or seed is provided. Infurther embodiments, the concentration/level of the at least onesequence that confers enhanced resistance to cyst nematodes (i.e., SEQID NO: 1, 2, 3, 4, or 5 or an active variant or fragment thereof) isincreased in a plant or plant part by at least 10%, 20%, 30%, 40%, 50%,60%, 70%, 80%, 90%, 100%, 200%, 500%, 1000%, 5000%, or 10,000% relativeto an appropriate control plant, plant part, or cell which did not havethe sequence. In still other embodiments, the level of the at least onesequence that confers enhanced resistance to cyst nematodes (i.e., SEQID NO: 1, 2, 3, 4, or 5 or an active variant or fragment thereof) in theplant or plant part is increased by 10, 20, 30, 40, 50, 60, 70, 80, 90,100 fold or more relative to an appropriate control plant, plant part,or cell which did not have the sequence. Such an increase in the levelof the at least one sequence that confers enhanced resistance to cystnematodes (i.e., SEQ ID NO: 1, 2, 3, 4, or 5 or an active variant orfragment thereof) can be achieved in a variety of ways including, forexample, by the expression of multiple copies of one or more of at leastone sequence that confers enhanced resistance to cyst nematodes (i.e.,SEQ ID NO: 1, 2, 3, 4, or 5 or an active variant or fragment thereof)and/or by employing a promoter to drive higher levels of expression ofone or more of the sequences.

In specific embodiments, at least one polynucleotide that confersenhanced resistance to cyst nematodes (i.e., SEQ ID NO: 1, 2, 3, 4, or 5or an active variant or fragment thereof or any combination thereof) isintroduced into the plant, plant cell, explant or plant part.Subsequently, a plant cell or plant having the introduced sequence isselected using methods known to those of skill in the art such as, butnot limited to, Southern blot analysis, DNA sequencing, PCR analysis, orphenotypic analysis. A plant or plant part altered or modified by theforegoing embodiments is grown under plant forming conditions for a timesufficient to modulate the concentration and/or activity of at least onesequence that confers enhanced resistance to cyst nematodes (i.e., SEQID NO: 1, 2, 3, 4, or 5 or an active variant or fragment thereof) in theplant.

VI. Sequence Comparisons

The following terms are used to describe the sequence relationshipsbetween two or more polynucleotides or polypeptides: “referencesequence”, “comparison window”, “sequence identity”, and, “percentsequence identity.”

As used herein, “reference sequence” is a defined sequence used as abasis for sequence comparison. A reference sequence may be a subset orthe entirety of a specified sequence; for example, as a segment of afull-length cDNA or gene sequence, or the complete cDNA or gene sequenceor protein sequence.

As used herein, “comparison window” makes reference to a contiguous andspecified segment of a polypeptide sequence, wherein the polypeptidesequence in the comparison window may comprise additions or deletions(i.e., gaps) compared to the reference sequence (which does not compriseadditions or deletions) for optimal alignment of the two polypeptides.Generally, the comparison window is at least 5, 10, 15, or 20 contiguousamino acid in length, or it can be 30, 40, 50, 100, or longer. Those ofskill in the art understand that to avoid a high similarity to areference sequence due to inclusion of gaps in the polypeptide sequencea gap penalty is typically introduced and is subtracted from the numberof matches.

Methods of alignment of sequences for comparison are well known in theart. Thus, the determination of percent sequence identity between anytwo sequences can be accomplished using a mathematical algorithm.Non-limiting examples of such mathematical algorithms are the algorithmof Myers and Miller (1988) CABIOS 4:11-17; the local alignment algorithmof Smith et al. (1981) Adv. Appl. Math. 2:482; the global alignmentalgorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443-453; thesearch-for-local alignment method of Pearson and Lipman (1988) Proc.Natl. Acad. Sci. 85:2444-2448; the algorithm of Karlin and Altschul(1990) Proc. Natl. Acad. Sci. USA 872264, modified as in Karlin andAltschul (1993) Proc. Natl. Acad. Sci. USA 90:5873-5877.

Computer implementations of these mathematical algorithms can beutilized for comparison of sequences to determine sequence identity.Such implementations include, but are not limited to: CLUSTAL in thePC/Gene program (available from Intelligenetics, Mountain View, Calif.);the ALIGN program (Version 2.0) and GAP, BESTFIT, BLAST, FASTA, andTFASTA in the GCG Wisconsin Genetics Software Package, Version 10(available from Accelrys Inc., 9685 Scranton Road, San Diego, Calif.,USA). Alignments using these programs can be performed using the defaultparameters. The CLUSTAL program is well described by Higgins et al.(1988) Gene 73:237-244 (1988); Higgins et al. (1989) CABIOS 5:151-153;Corpet et al. (1988) Nucleic Acids Res. 16:10881-90; Huang et al. (1992)CABIOS 8:155-65; and Pearson et al. (1994) Meth. Mol. Biol. 24:307-331.The ALIGN program is based on the algorithm of Myers and Miller (1988)supra. A PAM120 weight residue table, a gap length penalty of 12, and agap penalty of 4 can be used with the ALIGN program when comparing aminoacid sequences. The BLAST programs of Altschul et al (1990) J. Mol.Biol. 215:403 are based on the algorithm of Karlin and Altschul (1990)supra. BLAST nucleotide searches can be performed with the BLASTNprogram, score=100, wordlength=12, to obtain nucleotide sequenceshomologous to a nucleotide sequence encoding a protein of the invention.BLAST protein searches can be performed with the BLASTX program,score=50, wordlength=3, to obtain amino acid sequences homologous to aprotein or polypeptide of the invention. BLASTP protein searches can beperformed using default parameters. See,blast.ncbi.nlm.nih.gov/Blast.cgi.

To obtain gapped alignments for comparison purposes, Gapped BLAST (inBLAST 2.0) can be utilized as described in Altschul et al. (1997)Nucleic Acids Res. 25:3389. Alternatively, PSI-BLAST (in BLAST 2.0) canbe used to perform an iterated search that detects distant relationshipsbetween molecules. See Altschul et al. (1997) supra. When utilizingBLAST, Gapped BLAST, or PSI-BLAST, the default parameters of therespective programs (e.g., BLASTN for nucleotide sequences, BLASTP forproteins) can be used. See www.ncbi.nlm.nih.gov. Alignment may also beperformed manually by inspection.

Unless otherwise stated, sequence identity/similarity values providedherein refer to the value obtained using GAP Version 10 using thefollowing parameters: % identity and % similarity for a nucleotidesequence using GAP Weight of 50 and Length Weight of 3, and thenwsgapdna.cmp scoring matrix; % identity and % similarity for an aminoacid sequence using GAP Weight of 8 and Length Weight of 2, and theBLOSUM62 scoring matrix; or any equivalent program thereof. By“equivalent program” is intended any sequence comparison program that,for any two sequences in question, generates an alignment havingidentical nucleotide or amino acid residue matches and an identicalpercent sequence identity when compared to the corresponding alignmentgenerated by GAP Version 10.

GAP uses the algorithm of Needleman and Wunsch (1970) J. Mol. Biol.48:443-453, to find the alignment of two complete sequences thatmaximizes the number of matches and minimizes the number of gaps. GAPconsiders all possible alignments and gap positions and creates thealignment with the largest number of matched bases and the fewest gaps.It allows for the provision of a gap creation penalty and a gapextension penalty in units of matched bases. GAP must make a profit ofgap creation penalty number of matches for each gap it inserts. If a gapextension penalty greater than zero is chosen, GAP must, in addition,make a profit for each gap inserted of the length of the gap times thegap extension penalty. Default gap creation penalty values and gapextension penalty values in Version 10 of the GCG Wisconsin GeneticsSoftware Package for protein sequences are 8 and 2, respectively. Fornucleotide sequences the default gap creation penalty is 50 while thedefault gap extension penalty is 3. The gap creation and gap extensionpenalties can be expressed as an integer selected from the group ofintegers consisting of from 0 to 200. Thus, for example, the gapcreation and gap extension penalties can be 0, 1, 2, 3, 4, 5, 6, 7, 8,9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or greater.

GAP presents one member of the family of best alignments. There may bemany members of this family, but no other member has a better quality.GAP displays four figures of merit for alignments: Quality, Ratio,Identity, and Similarity. The Quality is the metric maximized in orderto align the sequences. Ratio is the quality divided by the number ofbases in the shorter segment. Percent Identity is the percent of thesymbols that actually match. Percent Similarity is the percent of thesymbols that are similar. Symbols that are across from gaps are ignored.A similarity is scored when the scoring matrix value for a pair ofsymbols is greater than or equal to 0.50, the similarity threshold. Thescoring matrix used in Version 10 of the GCG Wisconsin Genetics SoftwarePackage is BLOSUM62 (see Henikoff and Henikoff (1989) Proc. Nail. Acad.Sci. USA 89:10915).

As used herein, “sequence identity” or “identity” in the context of twopolynucleotides or polypeptide sequences makes reference to the residuesin the two sequences that are the same when aligned for maximumcorrespondence over a specified comparison window. When percentage ofsequence identity is used in reference to proteins it is recognized thatresidue positions which are not identical often differ by conservativeamino acid substitutions, where amino acid residues are substituted forother amino acid residues with similar chemical properties (e.g., chargeor hydrophobicity). When sequences differ in conservative substitutions,the percent sequence identity may be adjusted upwards to correct for theconservative nature of the substitution. Sequences that differ by suchconservative substitutions are said to have “sequence similarity” or“similarity”. Means for making this adjustment are well known to thoseof skill in the art. Typically this involves scoring a conservativesubstitution as a partial rather than a full mismatch, therebyincreasing the percent sequence identity. Thus, for example, where anidentical amino acid is given a score of 1 and a non-conservativesubstitution is given a score of zero, a conservative substitution isgiven a score between zero and 1. The scoring of conservativesubstitutions is calculated, e.g., as implemented in the program PC/GENE(Intelligenetics, Mountain View, Calif.).

As used herein, “percent sequence identity” means the value determinedby comparing two optimally aligned sequences over a comparison window,wherein the portion of the polynucleotide sequence in the comparisonwindow may comprise additions or deletions (i.e., gaps) as compared tothe reference sequence (which does not comprise additions or deletions)for optimal alignment of the two sequences. The percentage is calculatedby determining the number of positions at which the identical nucleicacid base or amino acid residue occurs in both sequences to yield thenumber of matched positions, dividing the number of matched positions bythe total number of positions in the window of comparison, andmultiplying the result by 100 to yield the percent sequence identity.

Two sequences are “optimally aligned” when they are aligned forsimilarity scoring using a defined amino acid substitution matrix (e.g.,BLOSUM62), gap existence penalty and gap extension penalty so as toarrive at the highest score possible for that pair of sequences. Aminoacids substitution matrices and their use in quantifying the similaritybetween two sequences are well-known in the art and described, e.g., inDayhoff et al. (1978) “A model of evolutionary change in proteins.” In“Atlas of Protein Sequence and Structure,” Vol. 5, Suppl. 3 (ed. M. O.Dayhoff), pp. 345-352. Natl. Biomed. Res. Found., Washington, D.C. andHenikoff et al. (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919. TheBLOSUM62 matrix (FIG. 10) is often used as a default scoringsubstitution matrix in sequence alignment protocols such as Gapped BLAST2.0. The gap existence penalty is imposed for the introduction of asingle amino acid gap in one of the aligned sequences, and the gapextension penalty is imposed for each additional empty amino acidposition inserted into an already opened gap. The gap existence penaltyis imposed for the introduction of a single amino acid gap in one of thealigned sequences, and the gap extension penalty is imposed for eachadditional empty amino acid position inserted into an already openedgap. The alignment is defined by the amino acids positions of eachsequence at which the alignment begins and ends, and optionally by theinsertion of a gap or multiple gaps in one or both sequences, so as toarrive at the highest possible score. While optimal alignment andscoring can be accomplished manually, the process is facilitated by theuse of a computer-implemented alignment algorithm, e.g., gapped BLAST2.0, described in Altschul et al, (1997) Nucleic Acids Res.25:3389-3402, and made available to the public at the National Centerfor Biotechnology Information Website (http://www.ncbi.nlm.nih.gov).Optimal alignments, including multiple alignments, can be preparedusing, e.g., PSI-BLAST, available through http://www.ncbi.nlm.nih.govand described by Altschul et al, (1997) Nucleic Acids Res. 25:3389-3402.

Non-Limiting Embodiments Include:

1. A method of identifying a first soybean plant or a first soybeangermplasm with enhanced resistance to cyst nematode comprising detectingin the genome of said first soybean plant or in the genome of said firstsoybean germplasm at least one marker allele associated with aduplication of a region within the rhg1 locus.

2. The method of embodiment 1, wherein the duplication of the regionwithin the rhg1 locus comprises a tandem duplication of the soybeangenome between about position GM18:1663448 and about positionGM18:1632228.

3. The method of embodiment 1, wherein the duplication of the regionwithin the rhg1 locus comprises the region as set forth in at least oneof SEQ ID NO: 1, 2, 3, 4, or 5.

4. The method of embodiment 1, wherein the marker allele comprises atleast one polymorphism set forth in Table 1 or Table 2.

5. The method of any one of embodiments 1-4, wherein the method furthercomprises selecting the first soybean plant or the first soybeangermplasm or a progeny thereof having the at least one marker allele.

6. The method of embodiment 5, further comprising crossing the selectedfirst soybean plant with a second soybean plant.

7. A method of identifying a first soybean plant or a first soybeangermplasm with enhanced resistance to cyst nematode comprising detectingin the genome of said first soybean plant or said first soybeangermplasm a duplication of a region within the rhg1 locus.

8. The method of embodiment 7, wherein the duplication of the regionwithin the rhg1 locus comprises a tandem duplication of the region ofthe soybean genome between about position GM18:1663448 and aboutposition GM18:1632228.

9. The method of embodiment 7, wherein the duplication of the regionwithin the rhg1 locus comprises the region as set forth in at least oneof SEQ ID NO: 1, 2, 3, 4, or 5.

10. The method of embodiment 7, wherein the duplication of the regionwithin the rhg1 locus comprises each of the regions as set forth in SEQID NO: 1, 2, 3, 4, and 5.

11. The method of embodiment 7, 8, 9 or 10, wherein said detectingcomprises quantitative PCR or other quantitative technique.

12. The method of any one of embodiments 7-11, wherein the methodfurther comprises selecting the first soybean plant, the first soybeangermplasm or a progeny thereof having the duplication in the region ofthe rhg1 locus.

13. The method of embodiment 12, further comprising crossing theselected first soybean plant with a second soybean plant.

14. A method of identifying a first soybean plant or a first soybeangermplasm with enhanced resistance to cyst nematode comprising detectingin said first soybean plant or in said first soybean germplasm anincreased copy number of at least one of SEQ ID NO: 1, 2, 3, 4, or 5 oran active variant or fragment thereof.

15. The method of embodiment 14, wherein said method comprises detectingan increased copy number of SEQ ID NOS: 1, 2, 3, 4 and 5 or an activevariant or fragment thereof.

16. The method of any one of embodiments 14-15, wherein the methodfurther comprises selecting the first soybean plant, the first soybeangermplasm or a progeny thereof having the increased copy number of atleast one of SEQ ID NO: 1, 2, 3, 4, or 5.

17. The method of embodiment 16, further comprising crossing theselected first soybean plant with a second soybean plant.

18. A method for identifying a first soybean plant or a first soybeangermplasm with enhanced resistance to cyst nematode comprising detectingin the genome of said first soybean plant or said first soybeangermplasm a DNA junction formed at the breakpoint of a duplicated regionwithin the rhg1 locus.

19. The method of embodiment 18, wherein the duplicated region withinthe rhg1 locus comprises a tandem duplication of the region of thesoybean genome between about position GM18:1663448 and about positionGM18:1632228.

20. The method of embodiment 18, wherein the DNA junction comprises thesequence set forth in any one of SEQ ID NOS: 6, 7, 8 or 9 or a fragmentthereof.

21. The method of embodiment 18, wherein detecting the novel DNAjunction comprises PCR amplification of the DNA junction formed at thebreakpoint of the duplicated region within the rhg1 locus.

22. The method of embodiment 21, wherein said PCR amplification employsthe primer pair set forth in SEQ ID NO: 14 and 15.

23. The method of embodiment 18, wherein detecting the DNA junctioncomprises DNA sequencing.

24. The method of any one of embodiments 18-23, wherein the methodfurther comprises selecting the first soybean plant, the first soybeangermplasm or a progeny thereof having the DNA junction formed at thebreakpoint of the duplicated region within the rhg1 locus.

25. The method of embodiment 24, further comprising crossing theselected first soybean plant with a second soybean plant.

26. A plant or plant cell comprising a heterologous polynucleotideoperably linked to a promoter active in the plant or plant cell, whereinsaid heterologous polynucleotide comprises:

a) the nucleotide sequence as set forth in any one of SEQ ID NO: 1, 2,3, 4, or 5, or any combination thereof; or,

b) the nucleotide sequence having at least 85% sequence identity to anyone of SEQ ID NO: 1, 2, 3, 4, or 5, or any combination thereof; whereinexpression of said heterologous polynucleotide enhances said plantsresistance to cyst nematode.

27. The plant or plant cell of embodiment 26, wherein said plant orplant cell is from a monocot.

28. The plant or plant cell of embodiment 27, wherein said monocot ismaize, wheat, rice, barley, sugarcane, sorghum, or rye.

29. The plant or plant cell of embodiment 26, wherein said plant orplant cell is from a dicot.

30. The plant or plant cell of embodiment 29, wherein the dicot isBrassica, sunflower, cotton, or alfalfa.

31. The plant or plant cell of embodiment 29, wherein the dicot issoybean.

32. A transgenic seed from the plant of any one of embodiments 26-31,wherein said transgenic seed comprise the heterologous polynucleotide.

33. A method to enhance resistance to cyst nematode in a plantcomprising introducing into a plant cell a heterologous polynucleotideoperably linked to a promoter active in the plant, wherein saidheterologous polynucleotide comprises:

a) a nucleotide sequence as set forth in any one of SEQ ID NO: 1, 2, 3,4, or 5, or any combination thereof; or,

b) a nucleotide sequence having at least 85% sequence identity to anyone of SEQ ID NO: 1, 2, 3, 4, or 5, or any combination thereof;

wherein expression of said heterologous polynucleotide enhances saidplants resistance to cyst nematode.

34. The method of embodiment 33, wherein said plant is a monocot.

35. The method of embodiment 34, wherein said monocot is maize, wheat,rice, barley, sugarcane, sorghum, or rye.

36. The method of embodiment 33, wherein said plant is a dicot.

37. The method of embodiment 36, wherein the dicot is Brassica,sunflower, cotton, or alfalfa.

38. The method of embodiment 36, wherein the dicot is soybean.

EXPERIMENTAL

The following example is offered to illustrate, but not to limit, theclaimed invention. It is understood that the examples and embodimentsdescribed herein are for illustrative purposes only, and persons skilledin the art will recognize various reagents or parameters that can bealtered without departing from the spirit of the invention or the scopeof the appended claims.

Example 1

We have observed a previously unexplained pattern of inheritedheterozygosity at the rhg1 locus within lines harboring the PI88788derived rhg1-b allele. Several SNP variants within the fine-mappedregion of rhg1-b are heterozygous across PI88788 derived resistant lines(FIG. 1). This observation led us to hypothesize that tandem duplicationand subsequent degeneration of paralogous sequence could be responsiblefor the observed heterozygosity at this locus.

In order to investigate the paralogous copy-count, we analyzed deepresequencing data from Peking, PI88788, and Lincoln. Visualization ofsequencing coverage at the rhg1 locus suggested a substantial increasein copy-number for Peking and PI88788 relative to Lincoln consistentwith duplication of nucleotide sequences within this locus (FIG. 2).Furthermore, paired-end sequencing reads at the outer extremes of theduplicated region exhibited discordant alignments consistent with tandemduplication at the locus. Taken together, these results suggest thattandem duplication of nucleotide sequences within the rhg1 locus.

Quantitative comparison of the sequencing depth within and adjacent tothe putative duplication was performed by normalizing PI88788 and Pekingcoverage to Lincoln in a moving window across the Rhg1 locus. Consistentwith the visualization, copy-number analysis indicated a 3-fold and9-fold increase in Peking and PI88788 respectively relative to Lincolnwithin the tandem duplication but not in the regions immediatelyadjacent (FIG. 3). These results suggest that rhg1 and rhg1-b representtwo distinct copy-variable alleles of an identical structural variantconsistent with different levels of phenotypic resistance.

To validate the observed copy-number qPCR assays were designed againstthe single-copy and variable-copy regions of the rhg1 locus. The resultsof these assays are consistent with sequencing data suggesting anapproximately 4-fold and 9-fold copy-number increase in Peking andPI88788 respectively relative to susceptible lines (FIG. 4).

In order to more precisely define the breakpoints of the duplication,paired-end sequencing reads with discordant alignments at the boundariesof the event were assembled, from which a 158 bp contig was recovered.The alignment of this contig to the reference is consistent with atandem duplication event with breakpoints at Gm18:1663448 andGm18:1632228 (FIG. 5). Interestingly, there is a 3-bp microhomologyshared between the breakpoints. To evaluate the correctness of theputative breakpoints, a series of PCR primers were designed to amplifyfragments which span the junction of the tandem duplication.

Amplification of PCR products across the breakpoints in Peking andPI88788 derived resistant lines but not susceptible genotypes areconsistent with the described tandem duplication (FIG. 6). Furthermore,the size of amplicons is consistent our expectations given the physicalpositions of putative breakpoints. In order to validate the targeted PCRproducts is in fact being observed in these lines, traditional Sangersequencing was performed for Peking and PI88788 amplicons (FIG. 7).

Taken together, our supporting results provide evidence that the rhg1locus of Peking and PI88788 harbors identical tandem duplication withtwo distinct copy-variable alleles. The observed increase in copy-numberfrom Peking to PI88788 can be explained by homologous recombinationbetween the paralogous copies and unequal crossing over. Assuming thatpositive selective pressure is acting on expansion of the copy-count atthe Rhg1 locus it follows that this tandem duplication may harbor a geneor genes whose dosage contributes favorably to the SCN resistancephenotype which has been fine mapped to this region.

This region contains four genes which are completely duplicated:Glyma18g2580, Glyma18g2590, Glyma18g2600, Glyma18g2610; and one genewhich is partially duplicated: Glyma18g2570. Striking anecdotal evidencesupports the involvement of one or more of these genes in SCNresistance: the coding sequences of two genes (Glyma18g2600 andGlyma18g2610) are unaffected by polymorphism in both sources.Interestingly, Glyma18g2600 is predicted to be a signal-mediatingscaffolding protein and annotated as a ‘predicted defense related gene’.

A useful by-product of our analysis has been the development of a qPCRassay which can be used to screen additional candidate material. Thisassay would be useful for identifying either the described rhg1 andrhg1-b copy-variable allele(s) or additional alleles which have not beendescribed.

Materials and Methods

Whole Genome Shotgun Sequencing

Whole genome shotgun sequencing libraries were prepared for Peking,P188788 and Lincoln by Mary Beatty Lab according to standard sequencingprotocols. Libraries were sequenced on the Illumina HiSeq instrument toan average depth of 17× genome sequencing coverage. Sequenced reads werealigned to the Soybean Genomic Assembly Glyma 1.01 (JGI) by bowtie2 andcompressed binary alignment/map files were generated by SAMtools.

Breakpoint Assembly from Sequencing Reads

Paired-end sequencing reads which aligned to the boundaries of theputative duplication were extracted from the BAM file by SAMtools andassembled using. The resulting contigs were blasted against the SoybeanGenomic Assembly Glyma1.01 (JGI) through the Pioneer BLAST Submissionand Retrieval page with settings Expect=0.01.

Copy-Number Analysis

Copy-number analysis was performed by calculating the sequencingcoverage in a moving window across the rhg1 fine-map region. Coveragewindows within each sample were normalized to the median sequencingdepth and then compared as a ratio of resistant to susceptible sources(Normalized coverage ratio). This ratio approximates the fold-differencebetween the two lines being compared.

qPCR Assay

PCR primers were designed against the single-copy and variable-copyregions of the rhg1 fine-mapped locus, six copy-variable and sixsingle-copy primer-pairs yielded useful information. Two sources ofPeking (Peking and 91Y90) and PI88788 (PI88788 and 93Y13) resistance andthree susceptible lines (Lincoln, Dunfield and CNS) were assayed in fourreplicates for all primer-pairs. The qPCR was completed using theSYBR-Green assay. Replicates were averaged and ΔΔCt analysis wasperformed pair-wise and averaged between every variable-copy andsingle-copy combination. The distribution of these results was plottedfor each pairwise comparison between Peking, P188788 and susceptible.

The results confirm that the duplicated portion of the genome isactually duplicated.

Example 2 Conditions for PCR Amplification of Breakpoint Sequences 94C/4 min 35 Cycles:

-   -   94 C/30 s    -   60 C/45 s    -   72 C/2 min

Optional Dissociation Stage:

-   -   72 C/5 min        Primers were designed using Primer3        (biocomplx.phibred.com/hu/primer3.html) and checked for        uniqueness using GPS        (bioprodlx.phibred.com/Primer_search/cgi-bin/primer_hit_form.cgi).

Product Size Range: 550-650 bps Default Settings

Conditions for qPCR Copy Assay

95 C/5 min 40 Cycles:

-   -   95 C/20 sec    -   60 C/45 sec

Optional Dissociation Stage:

-   -   95 C/15 sec    -   60 C/20 sec    -   95 C/15 sec        Primers were designed using Primer3        (biocomplx.phibred.com/hu/primer3.html) and checked for        uniqueness using GPS        (bioprodlx.phibred.com/Primer_search/cgi-bin/primer_hit_form.cgi).

Product Size Range: 50-200 bps Primer Size: 18-24 opt 20

Tm: 59-62° C. (Primer pairs within 1° C. of each other)

Default Settings Nuclear DNA Extraction Protocol

Adapted from Meizhong Luo and Rod Wing. An Improved Method for Plant BACLibrary Construction. Methods in Molecular Biology, vol. 236: PlantFunctional Genomics: Methods and Protocols

Materials Per Extraction:

5—Sheets 6″x6″ Miracloth

3—50 mL tubes

1—funnel

Orbital Shaker

Solutions: 2× Nuclear Isolation Buffer (NIB) (2 L)

20 mM Tris-HCl, pH 8.0 (40 mL)

20 mM EDTA, pH 8.0 (80 mL)

200 mM KCl (29.82 g)

1 M sucrose (684.6 g)

8 mM spermidine (4.1 g)

2 mM spermine (1.4 g)

Sterilize by filtration. Store at 4° C.

NOTE: Dilute with sterile water to prepare 1×NIB, NIBT, and NIBM

1×NIBT: 1×NIB with 10% Triton X-100 (make 3 mL per extraction)1×NIBM: 1×NIB with 0.1%13-mercaptoethanol (make 65 mL per extraction)

Protocol:

-   -   1. Grind 1.5 g of lyophilized tissue in paint shaker    -   2. Transfer the ground tissue into a 50 mL tube containing 45 mL        ice cold NIBM    -   3. Keep tube on ice for 15 min while shaking gently on orbital        shaker    -   4. Filter the homogenate through 3 layers of Miracloth into a        clean 50 mL tube. Squeeze the pellet to allow maximum recovery        of nuclei-containing solution. Use additional 10 mL NIBM to wash        the pellet and squeeze again.    -   5. Filter the nuclei-containing solution through 2 additional        layers of Miracloth into a clean 50 mL Tube.    -   6. Add 1:20 of NIBT (2.75 mL) to the nuclei-containing solution        and keep tube on ice for 15 min while shaking gently on orbital        shaker    -   7. Centrifuge the tubes at 4000 rpm at 4° C. for 20 min    -   8. Decant the supernatant and add 5 mL of NIBM to first tube.        Resuspend via vortex.    -   9. Centrifuge mixture at 4000 rpm at 4° C. for 15 min    -   10. Decant the supernatant and proceed to Urea Extraction    -   11. Add 5 mL 7M Urea Buffer—Resuspend    -   12. Add 10 uL RNAse—Incubate 37 C for 30 min    -   13. Add 5 mL 25:24:1 Chloroform-Phenol-Octanol-Rock for 10        min—Centrifuge 20 min at 4000 rpm    -   14. Transfer supernatant, add 450 uL NaOAc and 5 mL Isopropanol        to new tube    -   15. Spin 15 min at 4000 rpm. Decant Supernatant. Clean with 70%        ethanol.    -   16. Resuspend in 500 uL 10 mM Tris.

Example 3

Table 1 provides a list of polymorphic sites found within the rhg1locus. The chromosomal position is denoted in the first two columns.“Ref” denotes the nucleotide occurring in the reference soybean sample,and “ALT” denotes the nucleotide found in the soybean lines havingenhanced resistance to SCN. The presence of the nucleotide alteration inthe Peking and the P188788 lines is denoted in the last two columns.Table 2 provides a list of polymorphic sites found within the codingregions of the rhg1 locus, specifically polymorphisms in Glyma18g2580(SEQ ID NO: 3), Glyma18g2590 (SEQ ID NO: 2), Glyma18g2600 (SEQ ID NO:4),Glyma18g2610 (SEQ ID NO:5); and Glyma18g2570 (SEQ ID NO:1) are provided.Thus, any one marker or any combination of the polymorphisms set forthin Tables 1 and 2 can be used to aid in identifying and selectingsoybean plans with enhanced resistance to SCN.

TABLE 1 #CHROM POS REF ALT Peking Glyma18g2570 Gm18 1631156 G C Yes Gm181631449 C T Yes Gm18 1631761 G T Yes Gm18 1632227 A G Yes Gm18 1633532 AG Yes Gm18 1633629 T A Yes Gm18 1633700 G A Yes Glyma18g2580 Gm181636766 T C Yes Gm18 1638717 T C Yes Glyma18g2590 Gm18 1644011 C T YesGm18 1642236 T C Yes Gm18 1643324 C T Yes Gm18 1643225 C G Yes Gm181642307 C T Yes Gm18 1642848 G A Yes Gm18 1644076 G C Yes Gm18 1644089 GA Yes Gm18 1640963 C T Yes Gm18 1641208 G A Yes Gm18 1641800 C A YesGm18 1644974 C A Yes Gm18 1644525 T C Yes Gm18 1640581 C T Yes Gm181642672 C G Yes Gm18 1644577 T G Yes Gm18 1644493 C T Yes Gm18 1645218 AT Yes Gm18 1641442 A G Yes Glyma18g2600 Glyma18g2610 m18 1652723 T C Yes

TABLE 2 #CHROM POS REF ALT Peking PI88788 Gm18 1544945 T G Yes No Gm181735510 T A Yes No Gm18 1730855 A T Yes No Gm18 1511134 C A Yes Yes Gm181684932 A G Yes No Gm18 1706708 G A Yes No Gm18 1727903 C T Yes No Gm181589715 G T Yes Yes Gm18 1545209 T G Yes No Gm18 1768415 A G Yes YesGm18 1536883 T C Yes No Gm18 1681958 A T Yes Yes Gm18 1556781 T C Yes NoGm18 1716068 A G Yes Yes Gm18 1582195 C T Yes Yes Gm18 1571774 A G YesYes Gm18 1681493 A G Yes Yes Gm18 1556678 A G Yes Yes Gm18 1623900 C TYes Yes Gm18 1682230 T A Yes Yes Gm18 1560784 A G Yes No Gm18 1731656 GT Yes No Gm18 1725991 A G Yes No Gm18 1674291 G A Yes Yes Gm18 1533453 GA Yes No Gm18 1615080 A G Yes No Gm18 1572987 A C Yes Yes Gm18 1641442 AG Yes Yes Gm18 1768805 A G Yes No Gm18 1709751 G A Yes Yes Gm18 1549465G A Yes Yes Gm18 1649892 T A Yes Yes Gm18 1645218 A T Yes Yes Gm181626676 T C Yes Yes Gm18 1567661 C T Yes No Gm18 1703321 T A Yes No Gm181644493 C T Yes No Gm18 1644577 T G Yes No Gm18 1516872 G A Yes No Gm181547825 C G Yes Yes Gm18 1633532 A G Yes Yes Gm18 1642672 C G Yes YesGm18 1657506 C T Yes Yes Gm18 1644525 T C Yes No Gm18 1653661 T C YesYes Gm18 1652453 A G Yes Yes Gm18 1543932 T C Yes No Gm18 1601192 T CYes No Gm18 1597849 T C Yes Yes Gm18 1641800 C A Yes Yes Gm18 1654906 CA Yes Yes Gm18 1552732 T G Yes Yes Gm18 1552753 A G Yes No Gm18 1710311T C Yes Yes Gm18 1640963 C T Yes Yes Gm18 1644089 G A Yes Yes Gm181623024 T C Yes Yes Gm18 1770832 A G Yes Yes Gm18 1693289 C T Yes YesGm18 1644076 G C Yes Yes Gm18 1649630 T C Yes Yes Gm18 1642848 G A YesYes Gm18 1655593 T A Yes No Gm18 1656348 T A Yes Yes Gm18 1643225 C GYes No Gm18 1638717 T C Yes Yes Gm18 1636305 C T Yes Yes Gm18 1645359 TG Yes Yes Gm18 1654849 C T Yes Yes Gm18 1656044 G A Yes Yes Gm18 1653887C T Yes No Gm18 1534496 T C Yes No Gm18 1633700 G A Yes Yes Gm18 1649385A G Yes Yes Gm18 1644011 C T Yes Yes Gm18 1652235 G C Yes Yes Gm181633948 G A Yes Yes Gm18 1646008 A C Yes Yes Gm18 1635588 A T Yes YesGm18 1656898 C T Yes Yes Gm18 1649069 G T Yes Yes Gm18 1649293 G C YesYes Gm18 1716401 C T Yes Yes Gm18 1658284 A G Yes Yes Gm18 1662766 T CYes Yes Gm18 1660183 T C Yes Yes Gm18 1750228 G A Yes No Gm18 1658707 GA Yes Yes Gm18 1639589 C G Yes Yes Gm18 1650140 G T Yes Yes Gm18 1656394C T Yes Yes Gm18 1766174 T C Yes Yes Gm18 1657162 C T Yes Yes Gm181652401 T C Yes Yes Gm18 1633629 T A Yes Yes Gm18 1649328 T C Yes YesGm18 1640338 A C Yes Yes Gm18 1561036 A C Yes Yes Gm18 1547861 A G YesYes Gm18 1710204 G A Yes Yes Gm18 1656462 C G Yes Yes Gm18 1573221 A CYes Yes Gm18 1636766 T C Yes Yes Gm18 1560705 A C Yes Yes Gm18 1663181 CT Yes Yes Gm18 1572279 G A Yes Yes Gm18 1670005 G A Yes Yes Gm18 1562844A G Yes Yes Gm18 1624569 G T Yes Yes Gm18 1654119 G A Yes Yes Gm181620249 G C Yes Yes Gm18 1678344 G A Yes Yes Gm18 1629210 G A Yes YesGm18 1695886 T A Yes Yes Gm18 1584541 A G Yes Yes Gm18 1557165 C T YesYes Gm18 1630038 A G Yes Yes Gm18 1633840 G A Yes Yes Gm18 1615738 G AYes Yes Gm18 1633983 T C Yes Yes Gm18 1663907 C G Yes Yes Gm18 1617696 AG Yes Yes Gm18 1736136 T C Yes Yes Gm18 1641208 G A Yes Yes Gm18 1573317A G Yes Yes Gm18 1694129 C A Yes Yes Gm18 1635553 C G Yes Yes Gm181682250 T C Yes Yes Gm18 1597865 A T Yes Yes Gm18 1601551 A T Yes YesGm18 1682082 A G Yes Yes Gm18 1515595 A G Yes Yes Gm18 1681789 T C YesYes Gm18 1737550 A C Yes Yes Gm18 1617770 C T Yes Yes Gm18 1710334 A TYes Yes Gm18 1662755 A G Yes Yes Gm18 1628083 A T Yes Yes Gm18 1663051 GA Yes Yes Gm18 1580305 C T Yes Yes Gm18 1682035 A G Yes Yes Gm18 1681786T C Yes Yes Gm18 1712832 T C Yes Yes Gm18 1624435 T C Yes Yes Gm181681523 A G Yes Yes Gm18 1597206 A T Yes Yes Gm18 1626749 A T Yes YesGm18 1616538 T C Yes Yes Gm18 1634118 G A Yes Yes Gm18 1582363 C T YesYes Gm18 1662894 A G Yes Yes Gm18 1582357 G A Yes Yes Gm18 1634974 A GYes Yes Gm18 1514385 G A Yes Yes Gm18 1737519 G A Yes Yes Gm18 1622766 TA Yes Yes Gm18 1640292 C A Yes Yes Gm18 1712691 A C Yes Yes Gm18 1732766T A Yes Yes Gm18 1684013 C A Yes Yes Gm18 1774737 A T, C Yes Yes Gm181663143 A G Yes Yes Gm18 1674972 C T Yes Yes Gm18 1567332 A C Yes YesGm18 1628315 T C Yes Yes Gm18 1578714 A G Yes Yes Gm18 1772453 A G YesYes Gm18 1687196 G T Yes Yes Gm18 1635378 C T Yes Yes Gm18 1683953 A GYes Yes Gm18 1623482 A G Yes Yes Gm18 1577205 A C Yes Yes Gm18 1587173 CT Yes Yes Gm18 1583431 G C Yes Yes Gm18 1707243 C A Yes Yes Gm18 1616691G A Yes Yes Gm18 1698859 T A Yes Yes Gm18 1607885 C T Yes Yes Gm181589780 A T Yes Yes Gm18 1606158 G C Yes Yes Gm18 1627607 A C Yes YesGm18 1712035 A C Yes Yes Gm18 1678648 C T Yes Yes Gm18 1560517 G A YesYes Gm18 1547327 T C Yes Yes Gm18 1634907 A T Yes Yes Gm18 1677749 C GYes Yes Gm18 1661259 C G Yes Yes Gm18 1582351 A G Yes Yes Gm18 1652253 AC Yes Yes Gm18 1547718 T C Yes Yes Gm18 1709679 A T Yes Yes Gm18 1563924G T Yes Yes Gm18 1681852 T C Yes Yes Gm18 1622152 T A Yes Yes Gm181583859 G T Yes Yes Gm18 1640137 A C Yes Yes Gm18 1622144 C T Yes YesGm18 1713907 A G Yes Yes Gm18 1634260 C T Yes Yes Gm18 1762238 T C YesYes Gm18 1635093 A G Yes Yes Gm18 1679658 C G Yes Yes Gm18 1617198 A GYes Yes Gm18 1573060 C G Yes Yes Gm18 1556663 A G Yes Yes Gm18 1661239 TC Yes Yes Gm18 1707415 C A Yes Yes Gm18 1733374 T C Yes Yes Gm18 1577661T C Yes Yes Gm18 1733491 A T Yes Yes Gm18 1733561 A G Yes Yes Gm181600951 C T Yes Yes Gm18 1587518 C T Yes Yes Gm18 1713576 A G Yes YesGm18 1675064 G A Yes Yes Gm18 1662694 T C, G Yes Yes Gm18 1739680 C GYes Yes Gm18 1662810 G A Yes Yes Gm18 1629986 G A Yes Yes Gm18 1694779 GA Yes Yes Gm18 1734669 G T Yes Yes Gm18 1752663 C G Yes Yes Gm18 1578154T C Yes Yes Gm18 1739797 A T Yes Yes Gm18 1611303 A G Yes Yes Gm181598160 G A Yes Yes Gm18 1618456 C T Yes Yes Gm18 1663731 C A Yes YesGm18 1663298 A G Yes Yes Gm18 1567602 A G Yes Yes Gm18 1686008 T C YesYes Gm18 1652723 T C Yes Yes Gm18 1640151 G T Yes Yes Gm18 1687035 C TYes Yes Gm18 1709802 A T Yes Yes Gm18 1604611 C G Yes Yes Gm18 1631156 GC Yes Yes Gm18 1635001 C T Yes Yes Gm18 1697427 C T Yes Yes Gm18 1661128A G Yes Yes Gm18 1716159 C T Yes Yes Gm18 1582843 A G Yes Yes Gm181628644 A C Yes Yes Gm18 1745955 C G Yes Yes Gm18 1685024 A G Yes YesGm18 1710068 G A Yes Yes Gm18 1582860 G A Yes Yes Gm18 1708735 G C YesYes Gm18 1662970 T C Yes Yes Gm18 1745545 T C Yes Yes Gm18 1611422 G AYes Yes Gm18 1686939 G A Yes Yes Gm18 1634620 G A Yes Yes Gm18 1607624 AG Yes Yes Gm18 1670786 T C Yes Yes Gm18 1766605 T C Yes Yes Gm18 1687192A T Yes Yes Gm18 1589020 A T Yes Yes Gm18 1515631 C A Yes Yes Gm181584144 T A Yes Yes Gm18 1712751 T C Yes Yes Gm18 1559659 G A Yes YesGm18 1675070 G A Yes Yes Gm18 1623183 C T Yes Yes Gm18 1664115 G A YesYes Gm18 1694637 T A Yes Yes Gm18 1627783 G A Yes Yes Gm18 1547820 A CYes Yes Gm18 1677584 G A Yes Yes Gm18 1567986 A T Yes Yes Gm18 1627489 TA Yes Yes Gm18 1693992 C G Yes Yes Gm18 1615940 C T Yes Yes Gm18 1690566A G Yes Yes Gm18 1661088 A G Yes Yes Gm18 1713030 A G Yes Yes Gm181758565 A C Yes Yes Gm18 1661104 T C Yes Yes Gm18 1576682 G T Yes YesGm18 1629930 T C Yes Yes Gm18 1634626 G A Yes Yes Gm18 1709893 G A YesYes Gm18 1616511 T C Yes Yes Gm18 1683849 G A Yes Yes Gm18 1623157 T AYes Yes Gm18 1754980 C A Yes Yes Gm18 1615882 A G Yes Yes Gm18 1767968 TC Yes Yes Gm18 1753786 A C Yes Yes Gm18 1725443 G C Yes Yes Gm18 1725460A C Yes Yes Gm18 1598339 G T Yes Yes Gm18 1679368 C G Yes Yes Gm181772255 C T Yes Yes Gm18 1772222 G C Yes Yes Gm18 1703814 T G Yes YesGm18 1651950 A C Yes Yes Gm18 1652011 C A Yes Yes Gm18 1622659 A T YesYes Gm18 1681897 G A Yes Yes Gm18 1712103 T C Yes Yes Gm18 1662935 T CYes Yes Gm18 1663250 G A Yes Yes Gm18 1751218 G A Yes Yes Gm18 1690472 CG Yes Yes Gm18 1755475 C A Yes Yes Gm18 1755033 T C Yes Yes Gm18 1612760T A Yes Yes Gm18 1753857 G A Yes Yes Gm18 1757955 T C Yes Yes Gm181737465 T C Yes Yes Gm18 1571274 T C No Yes Gm18 1693079 A G No Yes Gm181567327 G A No Yes Gm18 1691381 A G No Yes Gm18 1613850 T A No Yes Gm181752445 A G No Yes Gm18 1710853 A T No Yes Gm18 1719339 C A No Yes Gm181616566 G A Yes Yes Gm18 1578462 A G No Yes Gm18 1565457 T G No Yes Gm181740769 A G No Yes Gm18 1759101 C T No Yes Gm18 1583949 A G No Yes Gm181696534 G A No Yes Gm18 1737883 C T No Yes Gm18 1749495 A G No Yes Gm181719159 A T No Yes Gm18 1609752 G C No Yes Gm18 1761478 G A No Yes Gm181605226 C A No Yes Gm18 1763650 C T No Yes Gm18 1708722 C T No Yes Gm181763643 T C No Yes Gm18 1718688 T C No Yes Gm18 1616062 T G No Yes Gm181651977 A T Yes Yes Gm18 1697857 C T No Yes Gm18 1735740 G T No Yes Gm181735487 G A No Yes Gm18 1748982 T C No Yes Gm18 1573328 G C No Yes Gm181607666 G A No Yes Gm18 1559603 C T No Yes Gm18 1716038 C T No Yes Gm181696266 G A No Yes Gm18 1608370 G T No Yes Gm18 1696077 G C No Yes Gm181671483 A G No Yes Gm18 1739504 T G No Yes Gm18 1740018 T A No Yes Gm181574247 A T No Yes Gm18 1608832 A G No Yes Gm18 1690011 G T No Yes Gm181683126 A G No Yes Gm18 1698255 A G No Yes Gm18 1703298 T C No Yes Gm181513861 T A No Yes Gm18 1694688 C G No Yes Gm18 1694436 T C No Yes Gm181749291 C G No Yes Gm18 1565592 G T No Yes Gm18 1693180 G A No Yes Gm181701212 C T No Yes Gm18 1772998 C A No Yes Gm18 1568478 G C No Yes Gm181715380 A T No Yes Gm18 1726316 T G No Yes Gm18 1759258 T C No Yes Gm181686081 A G No Yes Gm18 1696369 G A No Yes Gm18 1746679 A G No Yes Gm181696473 G C No Yes Gm18 1767466 G C No Yes Gm18 1749394 T A No Yes Gm181603821 C G No Yes Gm18 1751476 C T No Yes Gm18 1701594 C T No Yes Gm181663724 G A No Yes Gm18 1603800 A G No Yes Gm18 1691866 C G No Yes Gm181603653 A G No Yes Gm18 1767437 T C No Yes Gm18 1757019 A G No Yes Gm181710506 A G No Yes Gm18 1597089 C A Yes Yes Gm18 1767783 T C No Yes Gm181751428 A G No Yes Gm18 1639379 C T Yes Yes Gm18 1625454 G A Yes YesGm18 1699011 T C No Yes Gm18 1604559 A G No Yes Gm18 1601036 T G Yes YesGm18 1611740 A T Yes Yes Gm18 1745621 C T No Yes Gm18 1623426 C T YesYes Gm18 1761511 A T Yes Yes Gm18 1701944 T C Yes Yes Gm18 1585332 T GYes Yes Gm18 1587141 G T Yes Yes Gm18 1684856 T A No Yes Gm18 1607524 AC No Yes Gm18 1587643 G T Yes Yes Gm18 1747560 T G Yes Yes Gm18 1739095G A Yes Yes Gm18 1739077 C G Yes Yes Gm18 1600980 C T No Yes Gm181717706 G A Yes Yes Gm18 1629894 C T Yes Yes Gm18 1772204 A G Yes YesGm18 1563541 A G Yes Yes Gm18 1696180 C T No Yes Gm18 1696950 T C YesYes Gm18 1659777 C T Yes Yes Gm18 1628760 T C Yes Yes Gm18 1571487 T CYes Yes Gm18 1678883 G C No Yes Gm18 1558913 A G No Yes Gm18 1734249 A TYes Yes Gm18 1695342 G T No Yes Gm18 1627637 A G Yes Yes Gm18 1629751 CT Yes Yes Gm18 1673096 T G Yes Yes Gm18 1558334 T G No Yes Gm18 1701338A G No Yes Gm18 1755499 C T No Yes Gm18 1716152 A G No Yes Gm18 1748909A T No Yes Gm18 1646211 T A Yes Yes Gm18 1740029 G C No Yes Gm18 1738846C T Yes Yes Gm18 1774322 G T No Yes Gm18 1600945 T C Yes Yes Gm181718972 G T No Yes Gm18 1583764 C A Yes Yes Gm18 1686815 G T Yes YesGm18 1586097 T A Yes Yes Gm18 1749616 T A Yes Yes Gm18 1745477 T C NoYes Gm18 1691773 G A No Yes Gm18 1617005 A G No Yes Gm18 1731486 T C YesYes Gm18 1729770 A G Yes Yes Gm18 1597188 T A Yes Yes Gm18 1611710 G TYes Yes Gm18 1597307 C T Yes Yes Gm18 1707154 A G Yes Yes Gm18 1753536 TC No Yes Gm18 1685464 A T Yes Yes Gm18 1712512 C A No Yes Gm18 1603584 GA No Yes Gm18 1737864 C A Yes Yes Gm18 1755392 G C Yes Yes Gm18 1697683C T No Yes Gm18 1698898 C T No Yes Gm18 1664567 A T Yes Yes Gm18 1603220C T No Yes Gm18 1690438 G A No Yes Gm18 1616174 T C Yes Yes Gm18 1661328T C Yes Yes Gm18 1651056 G A Yes Yes Gm18 1603778 T C Yes Yes Gm181692400 G A Yes Yes Gm18 1648850 C T Yes Yes Gm18 1736100 A C Yes YesGm18 1631761 G T Yes Yes Gm18 1715338 C A Yes Yes Gm18 1628651 T A YesYes Gm18 1625573 G A Yes Yes Gm18 1603713 A G Yes Yes Gm18 1761449 C TNo Yes Gm18 1661356 A G Yes Yes Gm18 1608702 T A No Yes Gm18 1748303 A GNo Yes Gm18 1683857 A G Yes Yes Gm18 1772471 T C Yes Yes Gm18 1688738 AT No Yes Gm18 1703225 A G No Yes Gm18 1713308 G C No Yes Gm18 1755935 TG Yes Yes Gm18 1597320 T C Yes Yes Gm18 1612017 G A No Yes Gm18 1700809T C No Yes Gm18 1656979 G A Yes Yes Gm18 1599788 C T Yes Yes Gm181520606 C A Yes Yes Gm18 1700633 T C Yes Yes Gm18 1746656 C G No YesGm18 1622131 G A Yes Yes Gm18 1746362 A G No Yes Gm18 1697657 T A No YesGm18 1716072 G C No Yes Gm18 1565451 C T No Yes Gm18 1673499 C T No YesGm18 1624123 G A Yes Yes Gm18 1701279 C T No Yes Gm18 1625788 C T YesYes Gm18 1726582 T C Yes Yes Gm18 1581602 T C Yes Yes Gm18 1689381 T CNo Yes Gm18 1772437 A G Yes Yes Gm18 1629940 A T No Yes Gm18 1718590 G CYes Yes Gm18 1646019 C G Yes Yes Gm18 1686303 C T No Yes Gm18 1716309 CA No Yes Gm18 1566550 G C No Yes Gm18 1750655 C T Yes Yes Gm18 1768802 GA No Yes Gm18 1625527 T C Yes Yes Gm18 1620813 T G Yes Yes Gm18 1673273A T No Yes Gm18 1745785 G A No Yes Gm18 1698627 T C No Yes Gm18 1597566A T Yes Yes Gm18 1698566 A G No Yes Gm18 1754069 C T No Yes Gm18 1772404T C Yes Yes Gm18 1631449 C T Yes Yes Gm18 1626278 G A Yes Yes Gm181718002 G A No Yes Gm18 1584054 A G Yes No Gm18 1620585 T C Yes Yes Gm181663032 G A Yes Yes Gm18 1740267 T C No Yes Gm18 1662953 G A Yes YesGm18 1585543 G A Yes Yes Gm18 1615684 T G Yes Yes Gm18 1701321 T C NoYes Gm18 1748829 G C No Yes Gm18 1594426 A G Yes Yes Gm18 1751985 C G NoYes Gm18 1655195 C T Yes Yes Gm18 1705619 T A No Yes Gm18 1755105 A GYes Yes Gm18 1663534 G A No Yes Gm18 1679306 C T Yes Yes Gm18 1752225 TC No Yes Gm18 1759124 A G No Yes Gm18 1634714 A G No Yes Gm18 1625677 TC Yes Yes Gm18 1668348 A T No Yes Gm18 1705293 T C No Yes Gm18 1694764 TG No Yes Gm18 1597599 T A Yes Yes Gm18 1603952 G A No Yes Gm18 1599306 TC No Yes Gm18 1518036 T G Yes No Gm18 1597531 G C Yes Yes Gm18 1696238 TG No Yes Gm18 1745863 G A No Yes Gm18 1578538 T C No Yes Gm18 1639658 TC Yes Yes Gm18 1704849 C T No Yes Gm18 1582739 A G No Yes Gm18 1704867 AG No Yes Gm18 1545114 A G Yes No Gm18 1705325 A G No Yes Gm18 1703153 TC No Yes Gm18 1567685 C T Yes No Gm18 1547096 C T No Yes Gm18 1547940 AC Yes Yes Gm18 1700832 C T Yes Yes Gm18 1686599 A T Yes Yes Gm18 1673454C T No Yes Gm18 1510450 A T Yes No Gm18 1562453 C T No Yes Gm18 1692534A C Yes Yes Gm18 1685811 C G Yes Yes Gm18 1612354 A G No Yes Gm181678526 T A Yes Yes Gm18 1707433 G T No Yes Gm18 1696737 C T No Yes Gm181695572 G T No Yes Gm18 1658617 A G Yes Yes Gm18 1577708 G C Yes YesGm18 1515878 C T Yes Yes Gm18 1701304 T C No Yes Gm18 1562162 G A Yes NoGm18 1663620 T C Yes Yes Gm18 1690868 A G Yes Yes Gm18 1600193 T C YesYes Gm18 1650201 G A Yes Yes Gm18 1746695 G C Yes Yes Gm18 1604259 G ANo Yes Gm18 1689722 C G No Yes Gm18 1612360 A C No Yes Gm18 1710944 C TYes Yes Gm18 1611666 A G Yes Yes Gm18 1576719 G A No Yes Gm18 1560860 CT No Yes Gm18 1567133 G A Yes Yes Gm18 1603723 C G No Yes Gm18 1619991 AC Yes Yes Gm18 1767892 T A No Yes Gm18 1567183 C T Yes Yes Gm18 1676732T A Yes Yes Gm18 1753603 A C No Yes Gm18 1716247 G A No Yes Gm18 1681023T G Yes No Gm18 1693474 T A No Yes Gm18 1516891 G A Yes No Gm18 1719027T C No Yes Gm18 1604867 A C No Yes Gm18 1687349 G A Yes Yes Gm18 1684975C T Yes No Gm18 1514879 C A Yes No Gm18 1698112 T A No Yes Gm18 1606360C T No Yes Gm18 1702096 T C Yes Yes Gm18 1727375 C T No Yes Gm18 1661293G A Yes Yes Gm18 1612060 T G Yes Yes Gm18 1712907 T C No Yes Gm181714901 G A No Yes Gm18 1704995 A T No Yes Gm18 1604206 C T Yes Yes Gm181604186 T C Yes Yes Gm18 1676730 C A Yes Yes Gm18 1609704 T G Yes YesGm18 1731725 T C Yes No Gm18 1627802 G A Yes Yes Gm18 1659914 G A YesYes Gm18 1753820 A G Yes Yes Gm18 1604236 C T No Yes Gm18 1761960 C A NoYes Gm18 1645582 G C Yes Yes Gm18 1577745 C A Yes Yes Gm18 1698888 A TNo Yes Gm18 1704228 G T Yes Yes Gm18 1605467 G A No Yes Gm18 1707668 C TYes Yes Gm18 1627079 G T Yes Yes Gm18 1594961 G T No Yes Gm18 1567679 TC No Yes Gm18 1750622 C T No Yes Gm18 1663568 A G Yes Yes Gm18 1604054 GA No Yes Gm18 1604478 C T No Yes Gm18 1726584 A G No Yes Gm18 1626935 AT Yes Yes Gm18 1693038 C T Yes Yes Gm18 1761727 C T No Yes Gm18 1680414C T No Yes Gm18 1552799 C A Yes No Gm18 1734544 T C Yes Yes Gm18 1626400A C Yes Yes Gm18 1755415 C G Yes Yes Gm18 1604307 G A No Yes Gm181597401 C T No Yes Gm18 1704096 C T Yes Yes Gm18 1737706 A C No Yes Gm181649934 G A Yes No Gm18 1761957 G A No Yes Gm18 1568634 T A No Yes Gm181690926 G A No Yes Gm18 1639656 G A Yes Yes Gm18 1766218 C G Yes YesGm18 1726637 G A No Yes Gm18 1568509 A C No Yes Gm18 1626216 T C Yes YesGm18 1734457 T G Yes Yes Gm18 1656263 G C Yes Yes Gm18 1567931 A G NoYes Gm18 1701969 G T Yes Yes Gm18 1738049 A G No Yes Gm18 1599720 C G NoYes Gm18 1701818 T C No Yes Gm18 1572384 G T No Yes Gm18 1750742 G A YesYes Gm18 1577669 T G Yes Yes Gm18 1692117 G A Yes Yes Gm18 1716282 G CNo Yes Gm18 1714211 T C No Yes Gm18 1750557 C T No Yes Gm18 1581762 T CNo Yes Gm18 1594480 T C No Yes Gm18 1562660 G C No Yes Gm18 1677244 T CYes Yes Gm18 1708276 T C Yes Yes Gm18 1667354 T C Yes No Gm18 1707358 GT Yes Yes Gm18 1742720 G A No Yes Gm18 1711952 T C Yes No Gm18 1716080 CT No Yes Gm18 1563768 A C No Yes Gm18 1762265 G A No Yes Gm18 1567642 TA No Yes Gm18 1602219 G T No Yes Gm18 1655836 T C Yes Yes Gm18 1676145 AT Yes Yes Gm18 1603367 C T No Yes Gm18 1709043 A G Yes Yes Gm18 1760930T C Yes Yes Gm18 1533800 A G Yes No Gm18 1715191 G A No Yes Gm18 1655585A T Yes No Gm18 1603794 G A No Yes Gm18 1626263 T G Yes Yes Gm18 1604000C T No Yes Gm18 1692294 C T Yes Yes Gm18 1676737 C T Yes Yes Gm181619793 A G Yes Yes Gm18 1664369 A C Yes No Gm18 1560584 C A No Yes Gm181558551 T C Yes No Gm18 1757659 C T No Yes Gm18 1605056 T C Yes Yes Gm181718612 C A Yes Yes Gm18 1600162 C G No Yes Gm18 1559787 A C No Yes Gm181618051 A T Yes Yes Gm18 1772385 T A No Yes Gm18 1663479 A G Yes YesGm18 1734037 G T Yes Yes Gm18 1606094 C T Yes Yes Gm18 1682578 T A YesNo Gm18 1671225 T C No Yes Gm18 1759241 A G No Yes Gm18 1706862 A C NoYes Gm18 1581931 T G No Yes Gm18 1645811 A G Yes Yes Gm18 1665206 A TYes Yes Gm18 1705027 T G No Yes Gm18 1606777 G T No Yes Gm18 1703606 C TYes Yes Gm18 1616203 C A Yes Yes Gm18 1761649 G A Yes Yes Gm18 1630475 CA Yes Yes Gm18 1675849 A C No Yes Gm18 1772506 C A No Yes Gm18 1772430 GA No Yes Gm18 1761525 A C Yes Yes Gm18 1705064 C T No Yes Gm18 1716076 CT No Yes Gm18 1567659 G A No Yes Gm18 1585587 A G Yes Yes Gm18 1755633 AC Yes Yes Gm18 1509418 C T Yes No Gm18 1692913 C T No Yes Gm18 1546840 AG No Yes Gm18 1747766 A T Yes No Gm18 1567648 A G No Yes Gm18 1605559 CG, T Yes Yes Gm18 1518071 T C Yes No Gm18 1515940 T A Yes Yes Gm181679942 G T No Yes Gm18 1751117 T C Yes Yes Gm18 1569374 T C Yes YesGm18 1618502 C T Yes No Gm18 1583939 A T Yes Yes Gm18 1750705 A G YesYes Gm18 1750083 G A No Yes Gm18 1603857 C A, G Yes Yes Gm18 1691500 C TNo Yes Gm18 1719055 G A No Yes Gm18 1755101 T G Yes Yes Gm18 1755078 G AYes Yes Gm18 1629721 T A Yes Yes Gm18 1692085 C A Yes Yes Gm18 1712955 CT No Yes Gm18 1715284 C A No Yes Gm18 1739073 C T Yes Yes Gm18 1517392 GT Yes No Gm18 1750402 T G No Yes Gm18 1565704 T G No Yes Gm18 1761627 AG No Yes Gm18 1595360 A G No Yes Gm18 1769483 A T No Yes Gm18 1683590 TC Yes No Gm18 1688067 C T Yes No Gm18 1627771 T G Yes Yes Gm18 1566793 TC No Yes Gm18 1765893 G A Yes Yes Gm18 1586217 A T Yes Yes Gm18 1751280C A Yes Yes Gm18 1625548 G A Yes Yes Gm18 1537665 T A Yes No Gm181625263 T C Yes Yes Gm18 1724082 A G Yes No Gm18 1604183 G A Yes YesGm18 1768130 T A No Yes Gm18 1604653 C T No Yes Gm18 1565646 T G Yes NoGm18 1750842 T C No Yes Gm18 1661428 G A Yes Yes Gm18 1754530 G T No YesGm18 1568214 A G No Yes Gm18 1565225 G A Yes No Gm18 1699266 A T No YesGm18 1567665 C T Yes No Gm18 1510523 T C Yes No Gm18 1708391 C T Yes YesGm18 1663133 C T Yes No Gm18 1520736 G T Yes Yes Gm18 1660682 T A YesYes Gm18 1729347 T C Yes No Gm18 1690921 T C No Yes Gm18 1614447 G T NoYes Gm18 1559970 T G No Yes Gm18 1772369 C T No Yes Gm18 1758544 G A YesYes Gm18 1566823 A C Yes Yes Gm18 1639405 T A Yes Yes Gm18 1645759 T CYes Yes Gm18 1733766 T C No Yes Gm18 1566996 T C Yes No Gm18 1674853 C GYes No Gm18 1681070 G A Yes No Gm18 1546638 G C No Yes Gm18 1772329 G AYes Yes Gm18 1566846 A G No Yes Gm18 1662946 G T Yes Yes Gm18 1772418 TC No Yes Gm18 1761624 G T No Yes Gm18 1708283 T C Yes Yes Gm18 1625895 AG Yes Yes Gm18 1713174 C T No Yes Gm18 1769041 C A No Yes Gm18 1706019 GA No Yes Gm18 1663957 A T No Yes Gm18 1654787 C T Yes No Gm18 1757452 AC Yes Yes Gm18 1752933 A C Yes Yes Gm18 1716191 C G No Yes Gm18 1676018A C Yes Yes Gm18 1759526 C A No Yes Gm18 1644974 C A Yes No Gm18 1767510A G No Yes Gm18 1737892 C T No Yes Gm18 1719318 A G No Yes Gm18 1603771G C No Yes Gm18 1685613 G T Yes No Gm18 1567691 T G No Yes Gm18 1649371G A Yes No Gm18 1750798 A G No Yes Gm18 1708286 T C Yes Yes Gm18 1625409T C Yes Yes Gm18 1558144 A G Yes Yes Gm18 1521175 T G Yes No Gm181759517 G A No Yes Gm18 1604482 T A No Yes Gm18 1705451 T G No Yes Gm181684330 C T Yes No Gm18 1577684 T C Yes Yes Gm18 1515987 A G Yes YesGm18 1716081 C G No Yes Gm18 1601534 A G Yes No Gm18 1756949 C T No YesGm18 1572368 C T Yes No Gm18 1741366 T A No Yes Gm18 1611921 C T Yes YesGm18 1680099 A G Yes Yes Gm18 1766246 C G Yes Yes Gm18 1728146 C A YesNo Gm18 1750529 T C No Yes Gm18 1582158 T A No Yes Gm18 1562719 A T YesYes Gm18 1663114 A G Yes No Gm18 1650928 A T Yes Yes Gm18 1586334 A CYes Yes Gm18 1601614 A G Yes No Gm18 1568490 A C Yes No Gm18 1567049 G ANo Yes Gm18 1701792 A G No Yes Gm18 1625924 T C Yes Yes Gm18 1625923 A GYes Yes Gm18 1589032 C T Yes No Gm18 1663007 T C Yes Yes Gm18 1749588 GT No Yes Gm18 1719483 C T Yes Yes Gm18 1566922 C T Yes Yes Gm18 1511934T G Yes No Gm18 1681782 G C Yes No Gm18 1770509 A T No Yes Gm18 1643324C T Yes Yes Gm18 1766547 A T Yes Yes Gm18 1656769 T G Yes Yes Gm181517146 G C Yes No Gm18 1677273 T G Yes Yes Gm18 1681373 G A Yes No Gm181700730 G A Yes Yes Gm18 1757027 A C No Yes Gm18 1531862 A T Yes No Gm181684979 T G Yes No Gm18 1511990 G A Yes No Gm18 1679131 A G No Yes Gm181742215 C T No Yes Gm18 1629507 G T Yes Yes Gm18 1608498 A C Yes No Gm181551459 A G No Yes Gm18 1625660 C A Yes Yes Gm18 1718801 C T No Yes Gm181750514 A G No Yes Gm18 1688457 T A No Yes Gm18 1769820 T C No Yes Gm181774619 T A No Yes Gm18 1719095 G T No Yes Gm18 1645745 T A Yes No Gm181562155 A T Yes No Gm18 1509260 C G Yes No Gm18 1667481 A G Yes No Gm181750271 T C No Yes Gm18 1700835 T C No Yes Gm18 1702208 G A Yes Yes Gm181605983 A G Yes Yes Gm18 1751187 G C No Yes Gm18 1602244 C G Yes No Gm181605716 G A No Yes Gm18 1568963 G A Yes Yes Gm18 1757449 G A No Yes Gm181695383 T C No Yes Gm18 1547174 T A No Yes Gm18 1546699 G A No Yes Gm181539580 A C Yes No Gm18 1750508 T C No Yes Gm18 1640581 C T Yes No Gm181691759 G A No Yes Gm18 1750274 G C No Yes Gm18 1749539 T C No Yes Gm181654687 G C Yes No Gm18 1547286 T C Yes No Gm18 1605349 A T No Yes Gm181646148 G T Yes Yes Gm18 1721829 A T Yes No Gm18 1765585 G A Yes No Gm181763519 T A Yes No Gm18 1664713 G C Yes No Gm18 1546801 T A Yes Yes Gm181620185 C A Yes Yes Gm18 1569395 G A No Yes Gm18 1599686 T G No Yes Gm181754035 T C No Yes Gm18 1695131 C A No Yes Gm18 1605389 C A No Yes Gm181702044 C T No Yes Gm18 1568826 A G Yes Yes Gm18 1565858 T C Yes YesGm18 1599688 T C No Yes Gm18 1576291 C T No Yes Gm18 1600072 G C, A YesYes Gm18 1775058 G T No Yes Gm18 1691221 T G Yes Yes Gm18 1612200 A T NoYes Gm18 1584669 A G Yes Yes Gm18 1625658 C T Yes Yes Gm18 1567728 T CNo Yes Gm18 1532390 C T Yes No Gm18 1723032 T C Yes No Gm18 1566821 T CNo Yes Gm18 1671082 C T Yes No Gm18 1749602 G C No Yes Gm18 1766585 C TNo Yes Gm18 1701871 A G No Yes Gm18 1544585 T C Yes No Gm18 1709236 T AYes No Gm18 1748836 T C No Yes Gm18 1728528 G T No Yes Gm18 1700842 T GNo Yes Gm18 1582570 T C Yes Yes Gm18 1523663 T G Yes No Gm18 1770435 T ANo Yes Gm18 1719606 G C Yes Yes Gm18 1755274 C G Yes Yes Gm18 1755282 AG Yes Yes Gm18 1715336 G A No Yes Gm18 1566520 C T No Yes Gm18 1662682 AG Yes No Gm18 1569074 T C No Yes Gm18 1649212 A C Yes Yes Gm18 1634453 GA Yes Yes Gm18 1634543 T C Yes Yes Gm18 1577746 G A Yes Yes Gm18 1700722G A Yes Yes Gm18 1771045 G A Yes No Gm18 1600060 C G, A Yes Yes Gm181609397 C A Yes No Gm18 1692517 T A Yes Yes Gm18 1645954 G A Yes YesGm18 1762836 A C No Yes Gm18 1692147 C T Yes Yes Gm18 1543865 G C Yes NoGm18 1642236 T C Yes No Gm18 1707377 T C Yes No Gm18 1750037 A G No YesGm18 1718659 G A No Yes Gm18 1516270 A C Yes Yes Gm18 1692128 T C YesYes Gm18 1599872 T C Yes Yes Gm18 1695415 G T Yes Yes Gm18 1677168 C TYes Yes Gm18 1585055 T A No Yes Gm18 1606615 T C Yes Yes Gm18 1673470 AG No Yes Gm18 1754518 G A No Yes Gm18 1599907 A G Yes Yes Gm18 1605281 GA No Yes Gm18 1613051 C T Yes No Gm18 1551284 T A Yes Yes Gm18 1512482 AC Yes No Gm18 1625424 G A Yes Yes Gm18 1707759 T C Yes Yes Gm18 1766626C A No Yes Gm18 1733211 G A Yes No Gm18 1517001 A C Yes No Gm18 1680507T C Yes No Gm18 1750541 T C No Yes Gm18 1698479 A T Yes No Gm18 1775132T A Yes No Gm18 1569405 G T No Yes Gm18 1696511 C A Yes No Gm18 1709479T A Yes No Gm18 1750722 C T No Yes Gm18 1663573 A G No Yes Gm18 1769619T A Yes No Gm18 1654484 A G Yes No Gm18 1550024 T C Yes No Gm18 1716448A C No Yes Gm18 1772176 A G Yes Yes Gm18 1727343 A C Yes No Gm18 1598279A C No Yes Gm18 1757577 T G No Yes Gm18 1670333 T A Yes No Gm18 1691565A G No Yes Gm18 1625346 C T Yes Yes Gm18 1754005 T G No Yes Gm18 1692151C T Yes Yes Gm18 1692860 A G No Yes Gm18 1574545 G T No Yes Gm18 1541662G A Yes No Gm18 1704965 G A No Yes Gm18 1715146 T C Yes No Gm18 1692318T G Yes Yes Gm18 1567835 T G No Yes Gm18 1655408 G A Yes No Gm18 1598562T G No Yes Gm18 1699346 G A Yes Yes Gm18 1765117 G A No Yes Gm18 1731068G A Yes No Gm18 1663148 A G Yes No Gm18 1684542 T C Yes Yes Gm18 1707716T G Yes Yes Gm18 1733949 G A Yes Yes Gm18 1579270 A G Yes No Gm181634452 T C Yes Yes Gm18 1700689 C G No Yes Gm18 1692275 A G Yes YesGm18 1605946 A G Yes Yes Gm18 1605965 A C Yes Yes Gm18 1768110 A G NoYes Gm18 1668887 G A Yes No Gm18 1707714 A G Yes Yes Gm18 1605417 G A NoYes Gm18 1750318 G A No Yes Gm18 1521150 C T Yes No Gm18 1750049 G A NoYes Gm18 1567716 C T No Yes Gm18 1568548 T A No Yes Gm18 1642307 C T YesNo Gm18 1773908 A T Yes Yes Gm18 1707839 A C Yes Yes Gm18 1750766 C A NoYes Gm18 1677210 A G Yes Yes Gm18 1663785 C T Yes No Gm18 1702736 C T NoYes Gm18 1668441 A G Yes No Gm18 1605631 C T No Yes Gm18 1605957 C T YesYes Gm18 1605958 C T Yes Yes Gm18 1599684 G C Yes No Gm18 1710295 A CYes No Gm18 1750099 G T No Yes Gm18 1658170 T C Yes No Gm18 1768783 C AYes No Gm18 1712967 C T No Yes Gm18 1576881 C T No Yes Gm18 1600084 T CNo Yes Gm18 1740118 C T Yes No Gm18 1603741 T C No Yes Gm18 1517895 C TYes No Gm18 1763183 G A No Yes Gm18 1705929 A G No Yes Gm18 1707866 G AYes Yes Gm18 1750817 C T No Yes Gm18 1663014 G T Yes Yes Gm18 1560390 AC Yes No Gm18 1542189 A G Yes No Gm18 1728824 G A Yes No Gm18 1689805 TC Yes No Gm18 1700740 A G Yes Yes Gm18 1603750 T C No Yes Gm18 1538040 CT Yes No Gm18 1750890 A C No Yes Gm18 1761318 T C No Yes Gm18 1605853 CT Yes Yes Gm18 1703801 A T Yes Yes Gm18 1701930 A C Yes Yes Gm18 1679739T C Yes No Gm18 1725630 G C Yes No Gm18 1762610 T A No Yes Gm18 1701700A C No Yes Gm18 1663486 G A No Yes Gm18 1566446 G A Yes No Gm18 1757980T C Yes Yes Gm18 1744716 C T No Yes Gm18 1603235 C T No Yes Gm18 1657307C T Yes Yes Gm18 1765324 G A No Yes Gm18 1709488 A T Yes No Gm18 1751156C T Yes No Gm18 1707841 T G Yes Yes Gm18 1725815 G A Yes No Gm18 1762541N A Yes Yes Gm18 1599868 T C Yes Yes Gm18 1736297 C T Yes Yes Gm181592850 T A Yes Yes Gm18 1717352 G T Yes No Gm18 1540542 A G Yes No Gm181706842 C G Yes No Gm18 1605573 G A No Yes Gm18 1583772 G A Yes No Gm181511910 T G Yes No Gm18 1570009 A C Yes Yes Gm18 1700743 C T Yes YesGm18 1569093 A C No Yes Gm18 1645908 T A Yes Yes Gm18 1516459 G C Yes NoGm18 1545944 C A Yes No Gm18 1654681 T A Yes No Gm18 1567459 C A No YesGm18 1715501 G A Yes No Gm18 1564092 A T Yes No Gm18 1579201 G A Yes NoGm18 1539750 G A Yes No Gm18 1659829 C T Yes No Gm18 1612553 G A Yes NoGm18 1538114 A G Yes No Gm18 1762540 N A Yes Yes Gm18 1701861 A G No YesGm18 1764886 A G Yes Yes Gm18 1542804 G A Yes No Gm18 1717676 A G YesYes Gm18 1568958 T C No Yes Gm18 1700692 T C No Yes Gm18 1702741 T C YesNo Gm18 1700725 C T Yes Yes Gm18 1692272 A T Yes Yes Gm18 1600077 G A NoYes Gm18 1567770 C T No Yes Gm18 1531244 G A Yes No Gm18 1772523 G A YesNo Gm18 1592832 T C Yes Yes Gm18 1684187 T C Yes No Gm18 1750533 G T NoYes Gm18 1717672 C T Yes Yes Gm18 1567714 A G No Yes Gm18 1578727 G TYes No Gm18 1663064 A G Yes No Gm18 1688429 T C No Yes Gm18 1678548 T AYes Yes Gm18 1692562 A G Yes Yes Gm18 1750710 T C No Yes Gm18 1555210 CG Yes No Gm18 1566988 C T Yes No Gm18 1541254 T C Yes No Gm18 1702482 CT Yes Yes Gm18 1771385 T G Yes Yes Gm18 1582767 C T Yes Yes Gm18 1612042A G Yes Yes Gm18 1567449 T G No Yes Gm18 1545916 C T Yes No Gm18 1700984T A No Yes Gm18 1605993 G A Yes Yes Gm18 1599921 G A Yes Yes Gm181750871 G A No Yes Gm18 1599897 A G Yes Yes Gm18 1517411 C A Yes No Gm181750012 G A No Yes Gm18 1651606 A T Yes Yes Gm18 1692394 C T Yes YesGm18 1707082 G A Yes No Gm18 1692180 A G Yes Yes Gm18 1758510 G A No YesGm18 1531264 T C Yes No Gm18 1568870 T G No Yes Gm18 1763296 G T Yes NoGm18 1754546 T C No Yes Gm18 1738263 T A Yes Yes Gm18 1762539 N C YesYes Gm18 1516055 C T Yes Yes Gm18 1598101 G T Yes No Gm18 1568939 G A NoYes Gm18 1516449 A G Yes No Gm18 1509282 G C No Yes Gm18 1684242 A G YesNo Gm18 1568012 G A Yes Yes Gm18 1666930 A G Yes No Gm18 1516837 G A YesNo Gm18 1547341 C T Yes No Gm18 1603797 T C Yes No Gm18 1568784 C G NoYes Gm18 1692368 C T No Yes Gm18 1605938 T C Yes Yes Gm18 1766496 G A NoYes Gm18 1767775 T A No Yes Gm18 1605297 G A No Yes Gm18 1678365 A C YesNo Gm18 1659371 A G No Yes Gm18 1568820 G A Yes Yes Gm18 1566882 C T YesYes Gm18 1759307 G A Yes No Gm18 1717699 T A Yes Yes Gm18 1733287 T CYes Yes Gm18 1630447 T G Yes Yes Gm18 1736377 G A Yes Yes Gm18 1752898 TA Yes Yes Gm18 1568019 A T Yes Yes Gm18 1766342 G A No Yes Gm18 1749959G C No Yes Gm18 1749955 T C No Yes Gm18 1666561 T C Yes No Gm18 1600097A G No Yes Gm18 1572432 T C No Yes Gm18 1625392 T G Yes Yes Gm18 1663967G A Yes No Gm18 1707757 T C Yes Yes Gm18 1567788 C T No Yes Gm18 1725932C T Yes No Gm18 1662666 G A Yes No Gm18 1568868 C A No Yes Gm18 1750007C A No Yes Gm18 1663535 T C Yes No Gm18 1592769 T C Yes Yes Gm18 1605606A G No Yes Gm18 1692198 A G Yes Yes Gm18 1692240 C T Yes Yes Gm181685571 T A Yes No Gm18 1708232 C A Yes Yes Gm18 1759764 T G Yes YesGm18 1751112 G A No Yes Gm18 1677142 G T Yes Yes Gm18 1691976 C T YesYes Gm18 1663501 C T No Yes Gm18 1727330 T C Yes No Gm18 1765635 T C NoYes Gm18 1717310 G A No Yes Gm18 1759724 T C Yes Yes Gm18 1635035 G AYes Yes Gm18 1626986 T A Yes Yes Gm18 1762538 N T Yes Yes Gm18 1595321 TC Yes No Gm18 1566434 T C Yes No Gm18 1753018 G A Yes Yes Gm18 1569369 TC No Yes Gm18 1635376 C A Yes No Gm18 1600011 C T Yes No Gm18 1692566 AT Yes Yes Gm18 1692784 G A No Yes Gm18 1707915 C T Yes Yes Gm18 1702563C A Yes No Gm18 1624678 C T Yes No Gm18 1676981 T C Yes Yes Gm18 1729866C T Yes No Gm18 1717224 G A No Yes Gm18 1600033 C T Yes No Gm18 1715288T G Yes No Gm18 1693319 A C No Yes Gm18 1566930 G A Yes Yes Gm18 1762377N A Yes Yes Gm18 1684117 A T Yes Yes Gm18 1569146 C T Yes No Gm181570126 G C No Yes Gm18 1762537 N G Yes Yes Gm18 1569349 N T Yes YesGm18 1667726 T C Yes No Gm18 1517191 G A Yes No Gm18 1614960 A T Yes NoGm18 1726680 G T No Yes Gm18 1726068 G T Yes No Gm18 1669829 T C Yes NoGm18 1766704 A G No Yes Gm18 1715459 T C Yes No Gm18 1700956 T G Yes YesGm18 1750473 C T No Yes Gm18 1760838 N G Yes Yes Gm18 1756288 A T No YesGm18 1708549 G A Yes No Gm18 1711611 T G No Yes Gm18 1562884 A G Yes NoGm18 1538857 T C Yes No Gm18 1760837 N G Yes Yes Gm18 1717303 C T No YesGm18 1512555 A T Yes No Gm18 1749997 G A No Yes Gm18 1653945 C T Yes NoGm18 1569348 N T Yes Yes Gm18 1729673 A G Yes No Gm18 1568005 A G YesYes Gm18 1762378 N C Yes Yes Gm18 1569128 C T No Yes Gm18 1623625 A GYes Yes Gm18 1560088 C T Yes No Gm18 1692818 G A, C Yes Yes Gm18 1766676C T No Yes Gm18 1662124 A T Yes Yes Gm18 1592838 C T Yes Yes Gm181669151 T A Yes No Gm18 1619145 A G Yes No Gm18 1675126 G A Yes No Gm181707897 C T Yes Yes Gm18 1565888 C T Yes Yes Gm18 1766655 C A Yes YesGm18 1519508 C G Yes No Gm18 1666849 T A Yes No Gm18 1663623 C T Yes NoGm18 1701854 A G Yes No Gm18 1569347 N A Yes Yes Gm18 1520675 C T Yes NoGm18 1766798 T A No Yes Gm18 1700959 T C Yes Yes Gm18 1762379 N T YesYes Gm18 1708127 T C Yes Yes Gm18 1524498 G T Yes No Gm18 1680668 T GYes No Gm18 1545360 A G Yes No Gm18 1677459 A G Yes No Gm18 1687115 A TYes No Gm18 1650501 T A Yes No Gm18 1717349 A C No Yes Gm18 1700966 G ANo Yes Gm18 1674960 C T Yes No Gm18 1674957 T A Yes No Gm18 1623626 C TYes Yes Gm18 1563153 G T Yes No Gm18 1569346 N T Yes Yes Gm18 1541604 CA Yes No Gm18 1707790 G A Yes Yes Gm18 1600007 C T No Yes Gm18 1606554 GA No Yes Gm18 1765046 G C No Yes Gm18 1765048 G C No Yes Gm18 1751052 AG No Yes Gm18 1715256 A G Yes No Gm18 1684351 G A Yes No Gm18 1541073 CT Yes No Gm18 1759782 C A Yes Yes Gm18 1759784 C A Yes Yes Gm18 1762380N A Yes Yes Gm18 1765764 T C No Yes Gm18 1708244 A G No Yes Gm18 1654795T A Yes No Gm18 1716443 T A No Yes Gm18 1692787 T C No Yes Gm18 1708120C A Yes Yes Gm18 1569345 N A Yes Yes Gm18 1579346 T G Yes No Gm181765552 T C No Yes Gm18 1599864 G A Yes No Gm18 1561009 G A Yes No Gm181550213 T A Yes No Gm18 1679773 A G Yes No Gm18 1702269 A T Yes Yes Gm181740974 T A Yes Yes Gm18 1576454 G T Yes Yes Gm18 1599836 T G No YesGm18 1559151 A T Yes No Gm18 1599608 C T No Yes Gm18 1669541 T C Yes NoGm18 1669348 C T Yes Yes Gm18 1691780 A T No Yes Gm18 1565900 G A YesYes Gm18 1762381 N T Yes Yes Gm18 1762382 N T Yes Yes Gm18 1565926 A GYes Yes Gm18 1618118 T A Yes No Gm18 1754310 A G Yes No Gm18 1599717 G AYes No Gm18 1680693 G T Yes No Gm18 1566975 T C Yes Yes Gm18 1733286 A GYes Yes Gm18 1703713 A T Yes Yes Gm18 1565865 T G Yes Yes Gm18 1752043 TA Yes Yes Gm18 1766803 G A No Yes Gm18 1565931 A G Yes Yes Gm18 1690522G C Yes No Gm18 1634323 C T Yes No Gm18 1766736 A G No Yes Gm18 1554156T A Yes No Gm18 1762686 A G Yes No Gm18 1775071 G C No Yes Gm18 1762535N A Yes Yes Gm18 1762536 N A Yes Yes Gm18 1762383 N A Yes Yes Gm181599975 A G No Yes Gm18 1565917 T C Yes Yes Gm18 1700569 T G Yes YesGm18 1725538 T G Yes No Gm18 1751021 G A No Yes Gm18 1692901 A T Yes NoGm18 1669115 T C Yes Yes Gm18 1666527 T A Yes No Gm18 1669481 A T Yes NoGm18 1753738 T A Yes Yes Gm18 1685923 C T Yes No Gm18 1691942 G C No YesGm18 1753009 T C Yes Yes Gm18 1764965 T C Yes Yes Gm18 1548716 C T YesNo Gm18 1518206 A G Yes No Gm18 1566429 C T Yes No Gm18 1762695 C G YesNo Gm18 1752808 A T Yes No Gm18 1762533 N A Yes Yes Gm18 1762534 N G YesYes Gm18 1654800 T A Yes No Gm18 1552671 A G Yes No Gm18 1749975 T C NoYes Gm18 1538953 A C Yes No Gm18 1707115 A G Yes No Gm18 1766276 G A NoYes Gm18 1512962 G T Yes No Gm18 1560166 G A Yes No Gm18 1577691 T C YesNo Gm18 1749887 C A No Yes Gm18 1766748 A T No Yes Gm18 1751663 T A YesYes Gm18 1614300 T A Yes No Gm18 1765697 A C No Yes Gm18 1677110 C T YesYes Gm18 1708139 G A Yes Yes Gm18 1764424 T C Yes Yes Gm18 1765126 C TNo Yes Gm18 1759795 C A Yes Yes Gm18 1538142 A G Yes No Gm18 1606442 T AYes No Gm18 1600154 T C Yes Yes Gm18 1751134 T C No Yes Gm18 1750927 A GNo Yes Gm18 1763858 T C Yes Yes Gm18 1541357 G A Yes No Gm18 1565864 T CYes Yes Gm18 1569140 A T No Yes Gm18 1543480 C T Yes No Gm18 1565826 T ANo Yes Gm18 1568761 G A No Yes Gm18 1765272 T C No Yes Gm18 1759787 C AYes Yes Gm18 1725547 T C No Yes Gm18 1566947 C T Yes Yes Gm18 1765925 CA No Yes Gm18 1560043 T A Yes No Gm18 1760836 N T Yes Yes Gm18 1657183 GT Yes No Gm18 1706442 A T No Yes Gm18 1759811 C T Yes Yes Gm18 1765783 AG No Yes Gm18 1669584 C A Yes No Gm18 1663553 T C Yes No Gm18 1585768 TA Yes Yes Gm18 1550153 T A Yes No Gm18 1670034 T C Yes No Gm18 1764443 AG Yes Yes Gm18 1692545 A T Yes Yes Gm18 1569344 N G Yes Yes Gm18 1759791C A Yes Yes Gm18 1753351 C A Yes No Gm18 1662177 G A Yes Yes Gm181604031 A G Yes No Gm18 1568929 A G Yes Yes Gm18 1569343 N A Yes YesGm18 1765276 G T No Yes Gm18 1550179 G C Yes No Gm18 1765155 A G No YesGm18 1561190 T A Yes No Gm18 1751031 C T No Yes Gm18 1766272 G A No YesGm18 1568541 A T Yes No Gm18 1600017 G A No Yes Gm18 1600015 G A No YesGm18 1765977 C T No Yes Gm18 1577633 T G Yes No Gm18 1687822 G A Yes NoGm18 1751017 T C No Yes Gm18 1700363 A T Yes No Gm18 1763838 C T Yes YesGm18 1569341 N T Yes Yes Gm18 1569342 N A Yes Yes Gm18 1734691 C T YesNo Gm18 1576755 G A Yes No Gm18 1708143 C A Yes Yes Gm18 1706995 G A YesNo Gm18 1766728 G C No Yes Gm18 1569136 T C No Yes Gm18 1766283 A T NoYes Gm18 1765717 C T No Yes Gm18 1600085 C T Yes No Gm18 1560170 A T YesNo Gm18 1669190 G A Yes No Gm18 1706441 C T No Yes Gm18 1766726 G T NoYes Gm18 1765151 G A No Yes Gm18 1759789 C A Yes Yes Gm18 1708057 G TYes Yes Gm18 1612408 T A Yes No Gm18 1664020 G A Yes No Gm18 1766458 A CNo Yes Gm18 1763828 A G Yes Yes Gm18 1749980 C T No Yes Gm18 1561783 A GYes No Gm18 1554570 A C Yes No Gm18 1599841 C T Yes No Gm18 1759095 T GYes No Gm18 1570660 T C Yes No Gm18 1554430 G A Yes No Gm18 1606726 C TYes Yes Gm18 1663564 A G Yes No Gm18 1599993 G A No Yes Gm18 1707774 T CYes Yes Gm18 1516597 T C Yes No Gm18 1551775 T A Yes No Gm18 1555237 G AYes No Gm18 1766719 T C No Yes Gm18 1705753 G A No Yes Gm18 1581688 T AYes No Gm18 1565948 G T Yes Yes Gm18 1669557 G A Yes No Gm18 1758523 C TNo Yes Gm18 1561651 C T Yes No Gm18 1555277 T C Yes No Gm18 1699343 C TYes No Gm18 1692163 C T Yes Yes Gm18 1568727 T G No Yes Gm18 1622108 A TYes Yes Gm18 1599986 C T No Yes Gm18 1509119 T C Yes No Gm18 1765980 C TNo Yes Gm18 1765228 A G No Yes Gm18 1524257 T A Yes No Gm18 1634636 C TNo Yes Gm18 1550053 G T Yes No Gm18 1756096 G A Yes No Gm18 1762531 N CYes Yes Gm18 1762532 N A Yes Yes Gm18 1766399 G A No Yes Gm18 1569035 GT Yes Yes Gm18 1766390 C T No Yes Gm18 1632227 A G Yes Yes Gm18 1569962C T No Yes Gm18 1612073 A T No Yes Gm18 1750156 C T No Yes Gm18 1766716G A No Yes Gm18 1552165 A C Yes No Gm18 1628562 G C Yes No Gm18 1548918C G Yes No Gm18 1639650 T C Yes Yes Gm18 1612428 A G Yes No Gm18 1752840G C No Yes Gm18 1676925 T A Yes Yes Gm18 1554604 A G Yes No Gm18 1606053G A Yes No

TABLE 3 Summary of SEQ ID NOs SEQ ID Description 1 Glyma18g2570 2Glyma18g2590 3 Glyma18g2580 4 Glyma18g2600 5 Glyma18g2610 6 Breakpointamplicon of peking soybean 7 Breakpoint amplicon of peking soybean 8Breakpoint amplicon of pi88788 soybean 9 Breakpoint amplicon of pi88788soybean 10 Forward Primer Rhg1_(dark black/dark black) 11 Reverse primerRhg1_(dark black/dark black) 12 Forward Primer Rhg1_(light grey/lightgrey) 13 Reverse primer Rhg1_(light grey/light grey) 14 Forward PrimerRhg1_(dark black/light grey) 15 Reverse primer Rhg1_(dark black/lightgrey) 16 Rhg1_Seq* forward primer 17 Rhg1_Seq* reverse primer 18 Contigspanning the duplication breakpoint 19 Contig spanning the duplicationbreakpoint 20 Contig spanning the duplication breakpoint 21 Contigspanning the duplication breakpoint 22 Sequence of amplicon spanning thebreakpoint. 23 Sequence of breakpoint 24 Sequence of amplicon spanningthe breakpoint. 25 Sequence of breakpoint 26-49 Primers shown in FIG. 8

All publications and patent applications mentioned in the specificationare indicative of the level of those skilled in the art to which thisinvention pertains. All publications and patent applications are hereinincorporated by reference to the same extent as if each individualpublication or patent application was specifically and individuallyindicated to be incorporated by reference.

Although the foregoing invention has been described in some detail byway of illustration and example for purposes of clarity ofunderstanding, it will be obvious that certain changes and modificationsmay be practiced within the scope of the appended claims.

That which is claimed:
 1. A method of identifying a first soybean plant or a first soybean germplasm with enhanced resistance to cyst nematode comprising: (a) detecting in the genome of said first soybean plant or in the genome of said first soybean germplasm at least one marker allele associated with a duplication of a region within the rhg1 locus; (b) detecting in the genome of said first soybean plant or said first soybean germplasm a duplication of a region within the rhg1 locus; (c) detecting in said first soybean plant or in said first soybean germplasm an increased copy number of at least one of SEQ ID NO: 1, 2, 3, 4, or 5 or an active variant or fragment thereof; or (d) detecting in the genome of said first soybean plant or said first soybean germplasm a DNA junction formed at the breakpoint of a duplicated region within the rhg1 locus.
 2. The method of claim 1a, wherein the duplication of the region within the rhg1 locus comprises a tandem duplication of the soybean genome between about position GM18:1663448 and about position GM18:1632228.
 3. The method of claim 1a, wherein the duplication of the region within the rhg1 locus comprises the region as set forth in at least one of SEQ ID NO: 1, 2, 3, 4, or
 5. 4. The method of claim 1a, wherein the marker allele comprises at least one polymorphism set forth in Table 1 or Table
 2. 5. The method of claim 1a, wherein the method further comprises selecting the first soybean plant or the first soybean germplasm or a progeny thereof having the at least one marker allele.
 6. The method of claim 1b, wherein the duplication of the region within the rhg1 locus comprises a tandem duplication of the region of the soybean genome between about position GM18:1663448 and about position GM18:1632228.
 7. The method of claim 1b, wherein the duplication of the region within the rhg1 locus comprises the region as set forth in at least one of SEQ ID NO: 1, 2, 3, 4, or
 5. 8. The method of claim 1b, wherein the duplication of the region within the rhg1 locus comprises each of the regions as set forth in SEQ ID NO: 1, 2, 3, 4, and
 5. 9. The method of claim 1b, wherein said detecting comprises quantitative PCR or other quantitative technique.
 10. The method of claim 1b, wherein the method further comprises selecting the first soybean plant, the first soybean germplasm or a progeny thereof having the duplication in the region of the rhg1 locus.
 11. The method of claim 1c, wherein said method comprises detecting an increased copy number of SEQ ID NOS: 1, 2, 3, 4 and 5 or an active variant or fragment thereof.
 12. The method of claim 1c, wherein the method further comprises selecting the first soybean plant, the first soybean germplasm or a progeny thereof having the increased copy number of at least one of SEQ ID NO: 1, 2, 3, 4, or
 5. 13. The method of claim 1d, wherein the duplicated region within the rhg1 locus comprises a tandem duplication of the region of the soybean genome between about position GM18:1663448 and about position GM18:1632228.
 14. The method of claim 1d, wherein the DNA junction comprises the sequence set forth in any one of SEQ ID NOS: 6, 7, 8 or 9 or a fragment thereof.
 15. The method of claim 1d, wherein detecting the novel DNA junction comprises PCR amplification of the DNA junction formed at the breakpoint of the duplicated region within the rhg1 locus.
 16. The method of claim 18, wherein said PCR amplification employs the primer pair set forth in SEQ ID NO: 14 and
 15. 17. The method of claim 1d, wherein detecting the DNA junction comprises DNA sequencing.
 18. The method of claim 1d, wherein the method further comprises selecting the first soybean plant, the first soybean germplasm or a progeny thereof having the DNA junction formed at the breakpoint of the duplicated region within the rhg1 locus.
 19. The method of claim 1, further comprising crossing the selected first soybean plant with a second soybean plant.
 20. A plant or plant cell comprising a heterologous polynucleotide operably linked to a promoter active in the plant or plant cell, wherein said heterologous polynucleotide comprises: a) the nucleotide sequence as set forth in any one of SEQ ID NO: 1, 2, 3, 4, or 5, or any combination thereof; or, b) the nucleotide sequence having at least 85% sequence identity to any one of SEQ ID NO: 1, 2, 3, 4, or 5, or any combination thereof; wherein expression of said heterologous polynucleotide enhances said plants resistance to cyst nematode.
 21. The plant or plant cell of claim 20, wherein (a) said plant or plant cell is from a monocot; (b) said plant or plant cell is maize, wheat, rice, barley, sugarcane, sorghum, or rye; (c) said plant or plant cell is from a dicot; or, (d) said plant or plant cell is Brassica, sunflower, cotton, or alfalfa.
 22. A transgenic seed from the plant of claim 21, wherein said transgenic seed comprise the heterologous polynucleotide.
 23. A method to enhance resistance to cyst nematode in a plant comprising introducing into a plant cell a heterologous polynucleotide operably linked to a promoter active in the plant, wherein said heterologous polynucleotide comprises: a) a nucleotide sequence as set forth in any one of SEQ ID NO: 1, 2, 3, 4, or 5, or any combination thereof; or, b) a nucleotide sequence having at least 85% sequence identity to any one of SEQ ID NO: 1, 2, 3, 4, or 5, or any combination thereof; wherein expression of said heterologous polynucleotide enhances said plants resistance to cyst nematode.
 24. The method of claim 23, wherein (a) said plant or plant cell is from a monocot; (b) said plant or plant cell is maize, wheat, rice, barley, sugarcane, sorghum, or rye; (c) said plant or plant cell is from a dicot; or, (d) said plant or plant cell is Brassica, sunflower, cotton, or alfalfa. 